CN112955258A - Systems and methods for allergen detection - Google Patents

Abstract

The present disclosure relates to devices and systems for target detection in samples, in particular allergen detection in food samples. The allergen detection system comprises a sampler, a disposable analysis cartridge and a detection device with an optimized optical system. Allergen detection utilizes nucleic acid molecules as detection agents and detection probes.

Description

Systems and methods for allergen detection

Cross Reference to Related Applications

This application claims us provisional patent application No. 62/741,756 filed on 5.10.2018; and priority of U.S. provisional patent application No. 62/862,174 filed on 17.6.2019; the contents of each of the above applications are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to portable devices and systems for target detection in a sample (e.g. allergen detection in a food sample). The present disclosure also provides methods for detecting the presence and/or absence of a (of interest) molecule (e.g., allergen) of interest in a sample.

Background

Allergy (e.g. food allergy) is a common medical condition. It is estimated that up to 2% of adults and up to 8% of children (especially children under the age of three) in the united states suffer from food allergies (about 1500 million people), and it is believed that this prevalence is increasing. A portable device that enables people with food allergies to test their food and determine the allergen content immediately and accurately would be beneficial in providing informed (informad) decisions about whether or not to eat.

Researchers have attempted to develop suitable devices and methods to meet this need, for example, in U.S. patent No. 5,824,554 to McKay; U.S. patent application publication nos. 2008/0182339 and 8,617,903 to Jung et al; U.S. patent application publication No. 2010/0210033 to Scott et al; U.S. Pat. No. 7,527,765 to Royds; U.S. patent No. 9,201,068 to Suni et al; and those disclosed in U.S. patent No. 9,034,168 to Khattak and river. There remains a need for improved molecular detection techniques. There is also a need for devices and systems that can detect allergens of interest in less time, with higher sensitivity and specificity (specificity) and with less technical expertise (expertise) than devices used today.

The present disclosure provides a portable assembly and device for rapidly and accurately detecting an allergen in a sample by using aptamer-based Signal Polynucleotides (SPNs). SPN as a detection agent specifically binds to the allergen of interest, thereby forming SPN: a protein complex. These complexes are detected and measured by a detection sensor. The sensor that captures SPN can include a chip (e.g., a DNA chip) printed with nucleic acid molecules that hybridize to SPN. The detection system may comprise a separate sampler, a disposable cartridge/container for processing the sample and performing the detection assay, and a detector unit comprising an optical system for operating the detection and detection of the reaction signal. The detection agent (e.g., SPN) and the sensor (e.g., DNA chip) can be integrated into a disposable cartridge of the present disclosure. The cartridge, detector and detection sensor can also be used in other detection systems. Other capture agents, such as antibodies to allergen proteins, may also be used in the detection system of the present invention. Consumers may use such devices in non-clinical settings, such as in homes, restaurants, school restaurants, and food processing facilities.

Disclosure of Invention

The present disclosure provides systems, devices, disposable cartridges/containers, optical systems, and methods for detecting molecules of interest (e.g., allergens) in various types of samples, particularly food samples. The allergen detection device and system is portable and handheld.

One aspect of the present disclosure is a component for detecting a molecule of interest in a sample (e.g., an allergen in a food sample). The assembly includes an analysis cartridge configured to receive a sample for processing to a state that allows for interaction of a molecule of interest with a detection agent (engage in). The assembly comprises a detector unit configured to receive an analysis cartridge in a configuration that allows detection of interaction of the molecule of interest with the detection agent by a detection mechanism housed by the detector unit. The interaction triggers a visual indication on the detector unit that indicates the presence or absence of the molecule of interest in the sample. The detector unit may be removably connected to the analysis cartridge.

In some embodiments, the assembly may further comprise a separate sampler configured to collect the sample to detect the molecule of interest in the sample. In some embodiments, the sampler is a food corer. The corer is operatively connected to the analysis cartridge to transfer the collected sample to the cartridge.

In some embodiments, the assay cartridge is disposable and is configured to detect a particular molecule of interest, such as an allergen. In other embodiments, the analysis cartridge may be configured to detect a plurality of molecules of interest, such as a set of allergens, in a sample.

In some embodiments, the analysis cartridge comprises a homogenizer (homogen) configured to produce a homogeneous sample, thereby releasing the molecule of interest from the matrix (matrix) of the sample into an extraction buffer (extraction buffer) optionally comprising a detection agent. The analysis cartridge further comprises a first conduit for transferring the homogeneous sample with or without the detection agent through the filter system to provide a filtrate comprising the molecule of interest or a complex of the molecule of interest and the detection agent, and a second conduit for transferring the filtrate to contact the filtrate with the detection probe to allow the detection agent to interact with the detection probe. The first and second conduits include a plurality of fluid paths that connect different portions of the conduits to transfer processed samples, buffers, filtrates, detection agents, waste, and other fluids.

In some embodiments, the analysis cartridge can further comprise a rotary valve system that provides a mechanism for controlling the transfer of sample and other fluidic components within the analysis cartridge. The rotary valve switching system can also be configured to provide a closed position to prevent fluid movement in the analysis cartridge.

In some embodiments, the homogenizer and rotary valve system may be powered by a motor located in the detector unit when the analysis cartridge is received by the detector unit.

In some embodiments, the assay cartridge comprises a plurality of chambers. The chambers are separate but connected for operation. As a non-limiting example, an analysis cartridge may include a sample processing chamber, a detection chamber, a waste chamber, and an optional buffer chamber. In some embodiments, the analysis cartridge may further comprise a separate filtrate chamber to hold the filtrate and optionally further concentrate the filtrate before it is transferred to the detection chamber. In some examples, the detection chamber includes a detection sensor and an optical window. The detection mechanism of the detector unit analyzes the detection reaction through the optical window to identify the interaction of the molecule of interest with the detection agent within the detection chamber.

In some embodiments, the assay cartridge comprises a detection sensor for measuring the interaction between the molecule of interest and the detection agent. The detection sensor is included in the detection chamber. In one non-limiting example, the detection sensor is a discrete substrate (substrate) that includes a plurality of fluidic channels and a detector chip area. The substrate is also known as a chip channel (chipannel), in which the fluidic channel is connected to the detector chip area. In some examples, the chip-type channel is a plastic substrate.

In some embodiments, the detector chip area within the chip-based channel includes at least one reaction panel and at least one control panel. In other embodiments, the detector chip area within a chip-based channel may include one reaction panel and two control panels. In other embodiments, the chip-based channel may include a plurality of reaction panels and a plurality of control panels. Optionally, the detector chip area further comprises one or more fiducial points (fiducial spots) that guide image processing by an imaging mechanism (e.g., camera) of the detector unit. Any suitable reference object may be provided as a reference mark (marker) for reference.

In some embodiments, the detector chip area within the chip-based channel comprises detection probe molecules immobilized (immobilize) on a reaction panel. The detection probe is configured to perform a probe interaction with the detection agent. Interaction of the molecule of interest with the detection agent prevents the detection agent from performing a probe interaction with the detection probe. The detector chip area within the chip-based channel may also include optically detectable control probe molecules immobilized on one or more control panels for normalizing the signal output measured by the detection mechanism.

In a preferred embodiment, the chip-based channel is a plastic chip, wherein the reaction panel is printed with nucleic acid-based detection probes comprising nucleic acid sequences complementary to the nucleic acid sequences of the detection agent, and wherein the control panel is printed with nucleic acid-based control probe molecules that do not bind to the molecule of interest or the detection agent.

The analysis cartridge can further comprise: a chamber storing a wash buffer for washing the detection chamber; and a waste chamber for receiving the out-flowing contents of the detection chamber after washing. In some embodiments, the series of bridged fluid conduits may include: (a) a fluid connection between the wash buffer chamber and the detection chamber; and (b) a fluid connection between the detection chamber and the waste chamber.

In some embodiments, the filter in the analysis cartridge is a filter assembly comprising a bulk filter and a membrane filter. The body filter may include a coarse filter (gross filter) and a depth filter. In some embodiments, the filter assembly may further comprise a filter cover that may lock the rotary valve.

In some embodiments, the molecule of interest and the detection agent in the homogeneous sample may be contacted with the detection agent prior to contacting the molecule of interest and the detection agent with the detector probe. The contacting of the molecule of interest with the detection agent may occur in the extraction buffer during homogenization, or in the filter during filtration, or in the filtrate chamber. In some embodiments, MgCl₂The deposits are pre-stored in the filter or filtrate chamber.

In some embodiments, the analysis cartridge can include a data chip unit configured to provide cartridge information.

In some embodiments, the assembly of the present disclosure includes a detector unit operatively connected to an analysis cartridge. In some embodiments, the detector unit of the assembly comprises a detection mechanism to measure a detection signal, i.e. the interaction between the detection agent and the detector probe. As a non-limiting example, the detection mechanism is an imaging system, such as a camera for fluorescence imaging.

In some embodiments, the detector unit of the assembly includes an outer housing that provides support for integrated components for operating the detection reaction of the detector unit and measuring the detection signal and receiving the analysis cartridge. According to the present disclosure, a means for operating a detection reaction and measuring a detection signal includes: motors for driving and controlling homogenization, and for controlling the rotary valve; a pump to drive and control fluid flow of the processed sample, filtrate, buffer and waste in the compartments of the analysis cartridge; the optical system is used for detecting and visualizing the detection result; and a display window.

In some embodiments, the optical system may include excitation optics and emission optics and an optical reader. The optical system is modified to detect signals from the detector chip area of the chip-based channel inside the cartridge.

In other embodiments, the optical system may include a camera sensor (e.g., a CCD camera and a sCMOS camera) to generate an image of the detection reaction of the detector chip area of the chip-based channel. These images are then processed to indicate the detection result.

In some embodiments, the detection component may include a user interface that is accessible and controllable through a software application. The software may be run by a software application on a personal device, such as a smart phone, tablet, personal computer, laptop, smart watch, and/or other device. In some cases, the software may be run by an internet browser. In some embodiments, the software may be connected to a remote local server called a cloud.

In one non-limiting embodiment of the present disclosure, the detection assembly comprises: an analysis cartridge configured as a disposable test cup or cup-shaped container; a detector unit including a note pad (pocket) for receiving the test cup; and an optional sampler. The disposable test cup or cup-like container may be configured as an analysis module in which a sample is processed and molecules of interest (e.g., allergens) in the test sample are detected by interaction with a detection agent.

In some embodiments, a disposable test cup or cup-like container comprises: a top cover configured to accept a sample and seal the cup or cup-shaped receptacle, wherein the top cover comprises a port for accepting the sample and at least one vent filter (break filter) for allowing air to enter; a body portion configured to process the sample to a state that allows interaction of the molecule of interest with the detection agent; and a bottom cover configured to be connected to the body portion of the cup, thereby forming a detection chamber having an optical window at the bottom of the detection cup and providing a connection surface to the detector unit. The exterior of the bottom cover includes a plurality of ports for connecting to a plurality of motors located in the detector unit for operating the homogenizer, rotary valve system and flow of fluid. The optical window of the detection chamber is connected to a detection mechanism in the detector unit. In some embodiments, the test cup or cup-shaped container further comprises a detection sensor, such as a transparent substrate having detection probes immobilized thereon. The transparent substrate is a chip-like channel comprising a detection chip region and nucleic acid-based probes immobilized thereon, and a fluid path.

In one non-limiting embodiment of the present disclosure, a disposable test cup comprises: (a) a first compartment having a homogenizer for receiving a sample and processing the sample; the homogenizer is configured to produce a homogenized sample, thereby releasing the molecule of interest from the matrix of the sample into the extraction buffer in the presence of the detection agent and allowing the molecule of interest in the sample to undergo an interaction with the detection agent; (b) a second compartment for contacting the filtrate comprising the molecule of interest and the detection agent with the detection probe; the second compartment comprises a chip-based channel comprising a plurality of fluidic channels and a detection chip area having detection probes immobilized thereon; (c) a conduit for transferring the homogenized sample and the detection agent through the filtration system to provide a filtrate containing the molecule of interest and the detection agent; (d) a rotary valve system configured to regulate transfer of the homogeneous sample and the detection agent through the filtration system, transfer of the filtrate to the second compartment, and transfer of the wash buffer to the second compartment, and transfer of effluent contents from the second compartment to the waste chamber; (e) a compartment for holding a wash buffer for washing the detection zone; and (f) a waste chamber for receiving the effluent contents of the detection chamber. In some examples, the detection probe is configured to perform a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from performing the probe interaction with the detection probe. Fluid pathways within the chip-based channels divert filtrate, contact the filtrate with detection probes immobilized on the chip area, and transfer effluent contents to a waste chamber.

In some embodiments, the cup top cover further comprises a layer for providing an identification label.

In some embodiments, portions of the disposable test cup are molded together to form an analysis module.

Another aspect of the present disclosure relates to a method for detecting the presence and/or absence of a molecule of interest in a sample, the method comprising the steps of: (a) collecting a sample suspected of containing the molecule of interest, (b) homogenizing the sample in an extraction buffer in the presence of a detection agent, thereby releasing the molecule of interest from the sample to interact with the detection agent comprising a fluorescent moiety, (c) filtering the homogenized sample containing the molecule of interest and the detection agent; (d) contacting the filtrate comprising the molecule of interest and the detection agent with a detection probe molecule that undergoes a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from undergoing a probe interaction with the detection probe; (e) washing the contact in step (d) with a wash buffer; (f) measuring a signal output from the probe interaction of the detection probe molecule and the detection agent; and (g) processing the detected signal and visualizing the interaction between the detection probe and the detection agent.

Molecules of interest may include, but are not limited to, proteins and variants or fragments thereof, nucleic acid molecules (e.g., DNA or RNA molecules) or variants thereof, lipids, sugars, and small molecules. In some embodiments, the molecule of interest may be a protein or variants and fragments thereof. In one example, the molecule of interest is an allergen, such as a food allergen. The detection agent may be an antibody or variant thereof, a nucleic acid molecule or variant thereof, or a small molecule. In some embodiments, the detection agent is a nucleic acid molecule that includes a nucleic acid sequence that binds to a molecule of interest. In one example, the nucleic acid-based detection agent is a Signaling Polynucleotide (SPN) derived from an aptamer that includes a core nucleic acid sequence that binds to a molecule of interest. SPN can also include a detectable moiety, such as a fluorescent moiety. Thus, the detection probe may comprise a complementary nucleic acid sequence that hybridizes to a free sequence of SPN.

Drawings

Fig. 1 is a perspective view of an embodiment of an inspection system according to the present disclosure, the inspection system comprising: a detection device 100 having an outer housing 101 and a port or receptacle 102 configured to hold a disposable cartridge 300; a separate food corer 200, as one example of a sampler; a disposable test cup 300, as an example of an analysis cartridge. Optionally, a lid (lid)103, an execute/action button 104 allowing the user to perform an allergen detection test, and a USB port 105 may be included.

Fig. 2A is an exploded perspective view of one embodiment of a food corer 200 as an example of a sampler.

Fig. 2B is a perspective view of sampler assembly 200.

Fig. 3A is a perspective view of an embodiment of a disposable test cup 300 that includes a top 310, a body 320, and a bottom 330.

Figure 3B is a cross-sectional view of the test cup 300 showing features inside the cup 300.

Fig. 3C is an exploded view of the disposable test cup 300.

Fig. 3D is a top (left) and bottom (right) perspective view of the top cover 312.

Fig. 3E is an exploded view of the cup top cover 311.

Fig. 3F is a top perspective view (left view) and a bottom perspective view (right view) of the cup 320.

Fig. 3G is a bottom perspective view of the bottom of the upper housing 320a (top view) shown in fig. 3C, and a top perspective view of the interior of the outer housing 320b (bottom view) shown in fig. 3C.

Fig. 3H is a bottom perspective view (left view) and a top perspective view (right view) of the cup bottom cover 337.

Fig. 3I is a bottom perspective view of the cup bottom surface after assembly of the base 330 and cup 320.

Fig. 4A is an exploded view of one embodiment of a filter assembly 325.

Fig. 4B is a cut-away perspective view of one embodiment of the filtrate chamber 322, which includes a bed chamber 431 for placement of the filter assembly 325, a collection trough (collection groove) 432, and a filtrate collection chamber 433.

Fig. 5A is a perspective view of an alternative embodiment of a cup 300.

Fig. 5B is an exploded view of the disposable test cup 300 of fig. 5A (filter 325 not shown).

Fig. 5C is a cut-away perspective view of the cup 300 of fig. 5A.

Fig. 6A is an exploded view of an alternative embodiment of the cup 300.

Fig. 6B is a top perspective view (right view) and a bottom perspective view (left view) of cup 320 of fig. 6A.

Fig. 6C is a bottom perspective view of the cup bottom 337 and the bottom of the cup 320 of fig. 6A.

Fig. 6D is an alternative embodiment of a filter assembly 325.

Fig. 6E is a cross-sectional view of the filter cover 621 when assembled with the rotary valve 350.

Fig. 6F is a perspective view of the rotary valve 350 (top view) and a bottom perspective view of the bottom of the rotary valve 350 (bottom view).

Fig. 6G is a bottom perspective view (top view) and a top perspective view (bottom view) of the cup bottom cover 337 shown in fig. 6A.

FIG. 7A is an exploded view of an alternative embodiment of the cup 300; cup 300 includes chip-style channel 710.

Fig. 7B is a perspective view of the chip-based channel 710 shown in fig. 7A.

Fig. 7C is a bottom perspective view of chip-based channel 710.

Fig. 7D is a bottom perspective view of an alternative embodiment of a chip-based channel 710.

Fig. 7E is an exploded view of an alternative embodiment of the cup 300.

Fig. 7F is an alternative embodiment of a bowl, with a filter gasketed 623 over-molded to the bowl.

Fig. 7G is an alternative embodiment of the rotary valve 350 shown in fig. 7E.

Fig. 7H is a cross-sectional view of the cup 320 shown in fig. 7E, showing the overmolded seal 713, which combines the multiple parts into a single part.

Fig. 7I is an alternative embodiment of a cup bottom cover 337 with a compression coil spring 721.

Fig. 7J is a perspective view of the cup bottom cover 337 shown in fig. 7I, showing the compression coil spring 721 at the bottom.

Fig. 7K is a perspective view of a sacrificial weld bead material (sacrificial weld bead material)722 in the bottom of cup 320 shown in fig. 7E.

Fig. 8A is a top perspective view of the bowl 320 showing features related to homogenization, filtration (F), washing (W1 and W2), and waste.

Fig. 8B is a schematic diagram showing the position of the rotary valve 350 during sample preparation and sample washing.

Fig. 8C is a diagram showing the flow of fluid inside the cup 300.

Fig. 9A is a perspective view of the device 100.

Fig. 9B is a top perspective view of device 100 without cover 103.

Fig. 10A is a longitudinal cross-sectional view of the device 100.

Fig. 10B is a transverse cross-sectional view of the device 100.

Fig. 11A is a valve motor 1020 and associated components for controlling the operation of the rotary valve 350.

Fig. 11B is a top perspective view of the output link 1020 associated with the motor.

FIG. 12A is a top perspective view of one embodiment of an optical system 1030.

Fig. 12B is a side view of the optical system 1030 of fig. 12A.

Fig. 13A is a diagram of the chip sensor 333 showing a test area and a control area.

Fig. 13B is a top view of optical system 1030 and chip 333, showing the reflectance that provides a fluorescence measurement of chip 333.

FIG. 13C is a perspective view of another embodiment of the chip sensor 333 or sensing region 333' of the chip-based channel 710, showing one reaction panel 1312, one control panel 1313 and two reference panels 1311.

Fig. 13D shows an exemplary pattern of probes in the reaction and control panels of the detection zones 333' of the chip-based channels 710.

Fig. 14A shows the optical assembly 1030 in a straight mode.

Fig. 14B shows the optical assembly 1030 in a folded mode.

Fig. 14C is a cut-away perspective view of one end of the apparatus 100 (the right side of fig. 10B), showing the emissive optics 1420, which includes lenses 1421, 1423 and filters 1422a and 1422B, placed within a stepped bore 1480 in the apparatus 100.

Fig. 15A is a perspective view of another embodiment of an optical system 1030 that includes excitation optics 1510, emission optics 1520, and a camera-based detector 1530.

Fig. 15B is a cross-sectional view of the optical component of fig. 15A when the optical system is configured inside the detection apparatus 100.

FIG. 16A is a histogram (histogram) showing the presence and absence of MgCl₂And MgCl₂Buffer comparison of solutions, MgCl₂Freeze-dried preparation (MgCl)₂Intensity of SPN in lysophilized formulation).

FIG. 16B shows MgCl deposited from a cotton filter (cotton filter) supported on a 1 micron grid (mesh)₂Percentage of magnesium recovered in the formulation.

Detailed Description

The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification will control.

Analytical devices are used to ensure that food safety has not yet reached the level at which it is guaranteed to be carried out. In particular, no portable device based on a simple but accurate, sensitive and fast detection scheme has been developed to detect a variety of known allergens. In the context of food safety control, one of the latest reviews (reviews) on aptamer-based assays suggests that, although a number of commercial analytical tools have been developed to detect allergens, most of them rely on immunoassays. It is further shown that the selection of aptamers for this composition is emerging (Amaya-Gonz a lez et al, Sensors 2013,13, 16292-.

The present disclosure provides detection assemblies and systems that can specifically detect low concentrations of allergens in a variety of food samples. The detection system and/or device of the present disclosure is a miniaturized, portable and hand-held product, intended to have a compact size, thereby enhancing its portability and fine (discreet) operation. A user may carry the detection system and apparatus of the present disclosure and perform a rapid and real-time test for the presence and/or absence of one or more allergens in a food sample prior to consumption of the food. The detection system and apparatus according to the present disclosure may be used by a user at any location, for example at home or in a restaurant. The test system and/or device displays the test results as standard readouts and any user can do the test as a brief instruction on how to operate the test system and device. One particular utility of the detection system is the simplicity and rapidity of the system. In general, the detection systems and components of the present disclosure can also be used to detect any molecule of interest (i.e., any target) in a sample; the molecule of interest may be a protein or variant thereof, a nucleic acid molecule (e.g., a DNA or RNA molecule) or variant thereof, a lipid, a sugar, a small molecule, or a cell.

In some embodiments, the detection system is configured for simple, fast, and sensitive one-step execution (one-step execution) of the introduction of the sample into the system. The system can complete the detection test in less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, or less than 4 minutes, or less than 3 minutes, or less than 2 minutes, or less than 1 minute. In some examples, the detection may be completed in approximately 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, or 15 seconds.

According to the present disclosure, the detection system may comprise a mechatronic building process integrating electronic, mechanical and computational engineering to implement and control the process of the target detection test, including but not limited to rechargeable or replaceable batteries, motor drivers for processing the test samples, pumps for controlling the flow of processed sample solutions and buffers in the cartridge, printed circuit boards, and connectors to couple and integrate different components for rapid allergen testing. The detection device of the present disclosure further includes: an optical system configured to detect the presence and concentration of a molecule of interest (e.g., an allergen) in a test sample and convert the detection signal into a readable signal; and a housing for supporting other parts of the detection device and integrating different parts into a functional product.

In some embodiments, the detection system is configured such that a disposable analysis cartridge (e.g., a disposable test cup or cup-shaped container) specific to one or more particular molecules of interest (e.g., allergens) is configured to receive and process a test sample and perform a detection test, all solutions being packaged therein. Thus, all solutions can be confined in a disposable analysis cartridge. As a non-limiting example, a disposable peanut test cup may be used by a user to detect peanuts in any food sample and discarded after testing. This may prevent cross-contamination when different allergen tests are performed using the same device. In some embodiments, a separate sampler for collecting a test sample is provided.

According to the present disclosure, a disposable analysis cartridge includes a detection agent that specifically binds to and identifies an allergen or molecule of interest. The detection agent can be, but is not limited to, an antibody or variant thereof, a nucleic acid molecule or variant thereof, and a small molecule. In some embodiments, the detection agent can be a nucleic acid molecule that includes a nucleic acid sequence that specifically binds to the molecule of interest. Nucleic acid-based detection agents may be aptamers and Signal Polynucleotides (SPNs) derived from aptamers that can recognize target molecules (e.g., allergens). In some embodiments, the SPN captures target molecules in the sample to form an SPN: a target complex. Another detection probe comprising a short nucleic acid sequence complementary to the SPN sequence can be used to anchor the SPN to a solid substrate for signal detection. In other embodiments, the detection agent and detection probe may be attached to a solid substrate, such as the surface of a magnetic particle, silica, agarose particle, polystyrene bead, glass surface, plastic chip, microwell, chip (e.g., microchip), and the like. It is within the scope of the present disclosure that such detection agents and detection probes may also be incorporated into any suitable detection systems and instruments for similar use.

Detection assembly and system

According to the present disclosure, a detection system or assembly for conducting a detection test of a molecule of interest (e.g., an allergen) in a sample comprises: at least one disposable analysis cartridge for processing the sample to a state that allows interaction of the molecule of interest with the detection agent; and a detector unit for detecting and visualizing the detection result (i.e. the interaction between the molecule of interest and the detection agent). Optionally, the detection system may further comprise at least one sampler for collecting the test sample. The sampler may be any tool that can be used to collect a portion of the test sample, such as a spoon. In some aspects, as discussed below, a specially designed sampler may be included into the present detection system. The exemplary embodiments described below illustrate these detection systems and components for detecting allergens in a sample.

Typically, the analysis cartridge is configured to receive a sample for processing to a state that allows interaction of the molecule of interest with the detection agent. The detector unit is configured to receive the analysis cartridge in a configuration that allows detection of interaction of the molecule of interest with the detection agent by a detection mechanism housed by the detector unit. The interaction triggers a visual indication on the detector unit that indicates the presence or absence of the molecule of interest in the sample. The detector unit may be removably connected to the analysis cartridge.

As shown in fig. 1, an embodiment of a detection system or assembly of the present disclosure includes: a detection device 100 configured to process a test sample, perform an allergen detection test, and detect the results of the detection test; a separate food corer 200, as one example of a sampler; and a disposable test cup 300 as an example of an analysis cartridge. The detection device 100 comprises an external housing unit 101, which provides support for the various components of the detection device 100. The port or receptacle 102 of the testing device 100 is configured for docking with a disposable test cup 300, while the lid 103 is included to open and close the instrument. The outer housing unit 101 also provides surface space for buttons that allow a user to operate the device. An execute/action button 104, which allows the user to perform an allergen detection test, and a USB port 105 may be included. Optionally, a power plug (not shown) may also be included. In the course of conducting the allergen detection test, the food coring device 200, with the sample contained therein, is inserted into the disposable test cup 300, and the disposable test cup 300 is inserted into the port 102 of the detection device 100 for detection.

Sampling device

Collecting a properly sized sample is an important step in performing allergen detection tests. In some embodiments of the present disclosure, a separate sampler for picking up and collecting a test sample (e.g., a food sample) is provided. In one aspect, a core-pack-plunger concept (spring-packer-recipient concept) for picking and collecting food samples is disclosed herein. Such a mechanism may measure and collect one or several sized portions of the test sample and provide pre-treatment steps such as cutting, grinding (abrading) and/or mixing to facilitate homogenization and extraction or release of allergen proteins from the test sample. The sampler may be operatively connected to the analysis cartridge and the detection device to transfer the test sample to the cartridge for sample processing. In accordance with the present disclosure, a single food corer 200 is constructed for obtaining different types of food samples and collecting appropriately sized portions of the test samples. In one example, the sample is a liquid sample. In another example, the sample is a solid sample.

As shown in fig. 2A, an embodiment of the food coring device 200 may include three portions: a plunger (plunger)210 at the distal end; a handle 220 configured for coupling with a corer 230; a corer 230 at the proximal end. The plunger 210 has: a distal portion provided with a corer top grip 211 (fig. 2A) at the distal end, the top grip 211 facilitating manipulation of the plunger 210 up and down; a plunger stop 212 intermediate the plunger body; and a seal 213 at the proximal end of the plunger body. The handle 220 may include a snap (snap fit)221 at a distal end and a protruding flat collar (flat collar) connected to the corer 230 at a proximal end. In one embodiment, as shown in FIG. 2A, the protruding flat collar includes a flange 222. The corer 230 may include a proximal portion that is provided with a cutting edge 231 (fig. 2A) at the proximal-most end. The corer 230 is configured to cut and hold the collected sample for discharge into the disposable test cup 300.

In some embodiments, the distal end of the plunger 210 may comprise a push plate. The plate may be a flat plate in any shape. In a preferred embodiment, the push plate may be a rounded square with a flared surface. Furthermore, the shape of the rounded square constitutes an anti-roll feature when sampler 200 is on a flat surface. This feature may also keep the collected samples within the interior (i.e., sample region) of the corer 230 from contacting an external surface (e.g., a table when the sampler is located on the table).

In some embodiments, the protruding flat collar may be configured as a small ring, rib, or the like. Such protrusions (projections) may prevent the finger from sliding down into the sample area and also provide tactile orientation. As one non-limiting example, the protruding flat collar is a small circular ring.

In one embodiment, the plunger 210 may be inserted inside the corer 230, wherein the proximal end of the plunger 210 may protrude from the corer 230 to directly contact the test sample and pick up a sized portion of the test sample along with the cutting edge 231 of the corer 230 (fig. 2B). According to the present disclosure, the plunger 210 is used to expel the sampled food from the corer 230 into the disposable test cup 300, and also to carry some food (e.g., liquid and creamy food) into the corer 230. By interacting with the catch 221, the features of the plunger stop 212 may prevent the plunger 210 from being pulled back too far or out of the corer body 230 during sampling. The seal 213 at the proximal-most end of the plunger 210 may maintain an airtight seal to draw liquid into the corer 230 by pulling the plunger 210 back. In some embodiments, the plunger 210 may be provided with other types of seals (including molded features) or mechanical seals. Handle 220 is configured to allow a user to hold the coring component of sampler 200. For example, the skirt (skert) 222 provides a means for the user to operate the food sampler 200, push the corer 230 downward, and drive the corer 230 into the collected food sample.

In some embodiments, the plunger 210 may include markings (markers) to provide additional guidance to the user indicating the position of the plunger within the corer and its position relative to the minimum sampling line and the maximum sampling line. In some embodiments, lines indicating the minimum and maximum amounts of sample to be collected are added to the exterior of the corer 230. The user can correct the size of the (correct) sampling compartment by adjusting the minimum and maximum lines.

In some embodiments, the cutting edge 231 may be configured to pre-process the collected sample, allowing the sampled food to be cored by pushing, twisting, and/or cutting. The cutting edge 231 may cut a portion from the test sample. As some non-limiting examples, the cutting edge 231 may be a flat edge, a sharp edge, a serrated edge with different numbers of teeth, a sharp serrated edge, and a thin-walled edge. In other versions, the inner diameter of the corer 230 ranges from about 5.5mm to 7.5 mm. Preferably, the inner diameter of the corer 230 may be about 6.0mm to about 6.5 mm. The inner diameter of the corer 230 may be 6.0mm, 6.1mm, 6.2mm, 6.3mm, 6.4mm, 6.5mm, 6.6mm, 6.7mm, 6.8mm, 6.9mm, or 7.0 mm. The size of the corer 230 is optimized so that the user collects the correct number of test samples (e.g., 1.0 grams to 0.5 grams).

The various portions of the food coring apparatus 200 may be configured in any shape that is easy to handle, such as triangular, square, octagonal, circular, oval, and the like.

In some embodiments, the plunger 210 and other portions of the sampler may be different colors. As one non-limiting example, the plunger may be green and the corer may be transparent. The increased contrast (contrast) makes the position of the plunger relative to the sampler clearly visible. In other embodiments, the food coring apparatus 200 may be further provided with a means for weighing the picked test sample, such as a spring, a scale, or equivalents thereof. As one non-limiting example, the food corer 200 may be provided with a weighing tension module.

The food corer 200 may be made of a plastic material including, but not limited to, Polycarbonate (PC), Polystyrene (PS), poly (methyl methacrylate) (PMMA), Polyester (PET), polypropylene (PP), High Density Polyethylene (HDPE), polyvinyl chloride (PVC), thermoplastic elastomer (TPE), thermoplastic urethane (TPU), acetal (POM), Polytetrafluoroethylene (PTFE), or any polymer and combinations thereof.

In some embodiments, the sampler may be further configured for convenient use by a user. For example, the handle 220 may include a textured surface to create a better visual and tactile differentiation between the grip region and the sample region to communicate to the user where to hold the sampler 200.

The sampler (e.g., corer 200) may be operatively associated with an analysis cartridge (e.g., disposable cup 300) and/or a detection device (e.g., device 100). Optionally, the sampler may include an interface for connecting to the cartridge. Optionally, a shroud may be positioned on the proximal end of the sampler. Sampler 200 may also include a sensor positioned with sampler 200 to detect the presence of a sample in the sampler.

Disposable analysis cartridge

In some embodiments, the present disclosure provides an assay cartridge or container. As used herein, the terms "cartridge", "container" and "test cup" are used interchangeably. The analysis cartridge is configured for performing a detection test. As used herein, an analysis cartridge is also referred to as an analysis module. The assay cartridge is disposable and is for a particular allergen or a particular group of allergens (e.g., a tree nut allergen). The disposable assay cartridge is configured for processing a test sample to a state that allows interaction of the allergen of interest with a detection agent, e.g., dissociation of a food sample and allergen protein extraction, filtration of food particles, storage of reaction solution/reagents and detection agent, capture of the allergen of interest using detection agent (e.g., antibodies and nucleic acid molecules that specifically bind to allergen proteins). In one embodiment, the detection agent is a nucleic acid molecule, such as an aptamer and/or an SPN derived from an aptamer. In other embodiments, the detection agent can be an antibody specific for an allergen protein, such as an antibody specific for the peanut allergen protein Ara H1. In other embodiments, the detection agent may be any agent that can specifically recognize an allergen protein, such as chemical compounds, peptide aptamers and complexes that can specifically recognize an allergen protein. The present disclosure discusses food allergens as examples of molecules of interest that can be detected with the components of the present invention. One skilled in the art will appreciate that any target (i.e., molecule of interest) in a sample can be detected.

According to the present disclosure, at least one separate analysis cartridge is provided as part of the assembly. In other embodiments, the analysis cartridge can be configured for use with any other detection system.

In certain embodiments, disposable assay cartridges can only be used for allergen testing in samples and therefore can be made of low cost plastic materials, such as Acrylonitrile Butadiene Styrene (ABS), COC (cyclic olefin copolymer), COP (cyclic olefin polymer), transparent high density polyethylene (HOPE), Polycarbonate (PC), Polymethylmethacrylate (PMMA), polypropylene (PP), Polyvinylchloride (PVC), Polystyrene (PS), Polyester (PET), or other thermoplastics. Thus, the disposable analysis cartridge may be configured for any particular allergen of interest. In some embodiments, the disposable cartridges may be configured for only one particular allergen, which may avoid cross-contamination with other allergen reactions.

In some embodiments, the disposable cartridge is made of polypropylene (PP), COC (cyclic olefin copolymer), COP (cyclic olefin polymer), PMMA (poly (methyl methacrylate)), or Acrylonitrile Butadiene Styrene (ABS).

In other embodiments, the analysis cartridges may be configured to detect two or more different allergens in a test sample in parallel. In some aspects, the cartridge can be configured to detect two, three, four, five, six, seven, or eight different allergens in parallel. In one protocol, detecting the presence of multiple allergens simultaneously (e.g., two, three, four, five or more allergens) may produce a positive signal indicating which allergen is present. In another aspect, a system is provided for detecting the presence of an allergen, such as a peanut or tree nut, and generating a signal to indicate the presence of such allergen.

In some embodiments, the disposable analysis cartridge can be further configured to include a barcode that can store batch-specific parameters. The user can then read and store the stored information in any digital format.

In some embodiments, the analysis cartridge comprises a homogenizer configured to produce a homogenized sample, thereby releasing the molecule of interest from the matrix of the sample into an extraction buffer optionally comprising a detection agent. The analysis cartridge further comprises a first conduit for transferring the homogeneous sample with or without the detection agent through the filter system to provide a filtrate containing the molecule of interest and the detection agent, and a second conduit for transferring the filtrate to contact the filtrate with the detection probe to allow interaction of the detection agent with the detection probe. The first and second conduits include a plurality of fluid paths connecting different portions of the conduits for transferring processed samples, buffers, filtrates, waste and other fluids.

In some embodiments, the analysis cartridge can further include a rotary valve system that provides a mechanism for controlling the transfer of sample and other fluidic components (e.g., buffers, filtrates, and waste) in the analysis cartridge. The rotary valve switching system can be further configured to provide a closed position to prevent fluid movement in the analysis cartridge.

In some embodiments, the homogenizer and rotary valve system may be driven by a motor located in the detector unit, or any other motor mechanism provided by the connected detection device, when the analysis cartridge is received by the detector unit.

In some embodiments, the analysis cartridge may be configured to include one or more separate chambers, each configured for separate functions, such as sample reception, protein extraction, filtration, storage for buffers, reagents, and waste solutions, and detection reactions. The chambers are separate but connected for operation. For example, an analysis cartridge may include a sample processing chamber, a detection chamber, a waste chamber, and an optional buffer chamber. In some embodiments, the analysis cartridge may also include a separate filtrate chamber to hold the filtrate and optionally further concentrate the filtrate prior to transfer to the detection chamber. In some examples, the detection chamber may include a detection sensor and an optical window. The detection mechanism of the detector unit analyzes the detection reaction through the optical window to identify the interaction of the molecule of interest with the detection agent in the detection chamber. The detection window is operatively associated with a detection mechanism of the detection device.

In some embodiments, the assay cartridge comprises a detection sensor for measuring the interaction between the target molecule and the detection agent. The detection sensor is included in the detection chamber. In one non-limiting example, the detection sensor is a transparent substrate that includes a plurality of fluid channels and a detector chip area. The substrate is called a chip-type channel, in which the fluid channel and the detector chip area are connected. In some examples, the chip-type channel is a plastic substrate.

In some embodiments, the detector chip area within the chip-based channel includes at least one reaction panel and at least one control panel. In other embodiments, the detector chip area within a chip-based channel may include one reaction panel and two control panels. In other embodiments, the chip-based channel may include a plurality of reaction panels and a plurality of control panels. Optionally, the detector chip area of the chip-on-chip channel further comprises one or more reference points that guide image processing by an imaging mechanism (e.g., a camera) of the detector unit. Any suitable fiducial object may be provided as a fiducial marker for reference.

In some embodiments, the chip-based channels comprise detection probe molecules immobilized on a reaction panel in the detector chip area. The detection probe is configured to perform a probe interaction with the detection agent. The molecule of interest interacts with the detection agent, while preventing the detection agent from performing a probe interaction with the detection probe. The detector chip area within the chip-based channel may further comprise optically detectable control probe molecules immobilized on one or more control panels for normalizing the signal output measured by the detection mechanism. In some embodiments, the control probe molecule is a nucleic acid molecule that does not bind to the molecule of interest or the detection agent.

In a preferred embodiment, the chip-based channel is a plastic chip, wherein the reaction panel is printed with nucleic acid-based detection probes comprising nucleic acid sequences complementary to the nucleic acid sequences of the detection agent, and wherein the control panel is printed with nucleic acid-based control probe molecules that do not bind to the detection agent.

In some embodiments, detection agents, detection probes, buffers such as extraction buffers and wash buffers, and other components required to assemble the functional cartridge are included.

In some embodiments, the analysis cartridge can include a data chip unit configured to provide cartridge information.

In accordance with the present disclosure, an analysis cartridge can be configured in any suitable shape and size. Some exemplary embodiments of the assay cartridge are described below. The exemplary embodiments are not limiting on the design of the cartridge.

Exemplary embodiments of an analysis Cartridge

In some embodiments, a disposable analysis cartridge may be interpreted as a disposable test cup or cup-shaped container. The cup-shaped receptacle may comprise a plurality of compartments assembled into a functional analysis module. According to one embodiment of the test cup, as shown in FIG. 3A, the assembled disposable test cup 300 comprises three parts: a top (top of cup) 310, a body (body of cup) 320, and a bottom (bottom of cup) 330. The three parts are operatively connected to assemble the functional analysis module. The cup 300 further comprises: a homogenizing rotor 340 that rotates in two directions to homogenize the sample; a filter assembly 325 to filter the processed sample; a rotary valve 350 conceived to control the fluid flow inside the cup (fig. 3B); and fluid path(s) that deliver processed sample, mixture, filtrate, buffer, and reagents to different compartments of the test cup (not shown in fig. 3B).

The test cup body 320 can include multiple chambers. In one embodiment, as shown in fig. 3B, the test cup body 320 includes: a homogenizing chamber 321 comprising a food processing reservoir 801 (shown in figure 8C); a filtrate chamber 322 for collecting a sample solution after filtration through a filter (e.g., a 2-state filter 325 as shown in fig. 3B and 4A); a waste chamber 323 comprising a waste reservoir 803 (as shown in fig. 8C); and optionally a wash buffer storage chamber 324 comprising a wash liquid storage reservoir 802 (as shown in fig. 8C). Alternatively, the bowl 320 may include one or more separate washing compartments therein. In some embodiments, as shown in fig. 3B and 3H, a reaction chamber 331 (also referred to herein as a signal detection chamber) for the processed sample is included at the cup bottom 330. The reaction/detection chamber 331 may include a separate detection sensor (e.g., chip 333 shown in fig. 3B) with detection probes that react with the processed sample. All analytical reactions take place in the reaction/detection chamber 331 and produce a detectable signal (e.g., a fluorescent signal) therein. In some embodiments, for example, a detection agent (e.g., SPN) pre-stored in the homogenization chamber 321 may be pre-mixed with the test sample in the homogenization chamber 321, where the test sample is homogenized and the extracted allergen proteins react with the detection agent. The mixed reaction complex may be delivered to the filter 325 before being delivered to the reaction/detection chamber 331. In other examples, a detection agent (e.g., SPN) may be stored in the filtrate chamber 322. The processed sample is filtered through the filter assembly 325 and reacts with the detection agent stored in the filtrate chamber 322. The filtrate containing the molecule of interest and the detection agent is transferred to the detection chamber 331, wherein the detection agent interacts with detection probes immobilized on a sensor (e.g., chip 333) and a detection signal is measured.

In alternative embodiments, more than one buffer and reagent storage reservoir may be included in the buffer and reagent storage chamber 324. As one non-limiting example, the extraction buffer and the wash buffer may be stored separately in reservoirs within the buffer storage chamber 324.

Fig. 3C shows an exploded view of one exemplary embodiment of a disposable test cup 300, the disposable test cup 300 being configured to contain three main components, namely, a top portion 310, a housing or body 320, and a bottom portion 330. The cup top 310 may include a cup lid 311, a top cover 312, two or more vent filters 314 included to ensure that only air is introduced and fluid does not escape from the test cup 300. The cup 320 is made up of two separate parts: an upper housing 320a and an outer housing 320 b. The cup bottom assembly 330 includes a bottom cover 337 that clamps other components including a reaction chamber 331 (in fig. 3F and 3H), a detection sensor (i.e., a glass chip 333), and a chip gasket 334 that facilitates attachment of the glass chip 333 to the bottom of a dedicated sensor area 332 in the reaction chamber 331. In some embodiments, the processed sample mixer flows to reaction chamber 331 and reacts with a detection agent on chip 333 to generate a detectable signal. For example, chip 333 may be coated with oligonucleotide sequences to detect the presence of targets in the test sample. The bottom cover 337 also includes ports/nozzles (bit)340a for holding the homogenizing rotor 340 and ports/nozzles 350a for holding the rotary valve 350 (as shown in figure 3H). These nozzles provide means for coupling (link) the homogenizing rotor 340 and the rotary valve 350 to the motor of the inspection apparatus 100. In some embodiments, a rotor gasket 326 may be configured to the upper housing 320a to seal the rotor 340 to the housing 320 to avoid fluid leakage. In some embodiments, the bottom cover may further comprise a fluid path and an air channel.

In some embodiments, the cup may be further configured to include a bar code that may store batch-specific parameters. In one example, the barcode may be a data chip 335 that stores cup 300 specific parameters including information of the detection agent such as SPN (e.g., fluorophore label, target allergen, and intensity of SPN, etc.), expiration date, manufacturing information, and the like.

Fig. 3D further illustrates features of the top cover 312 of the cup shown in fig. 3A. A corer port 313 is included for receiving the food corer 200 to receive a picked-up test sample and transfer the sample to a sample processing chamber 321 (also referred to as a homogenization chamber). As one non-limiting example, the port 313 may be configured to receive a food coring apparatus 200 as shown in fig. 2B. The top cover 312 may also include at least one small hole (fig. 3D) for drawing air to flow the fluid. As one non-limiting example, the top portion may have two covers 311. As described above, the cover 311 may include two layers: a top cover 311a for sealing and labeling; and a bottom 311b for resealing during operation. As shown in fig. 3E, the second cover at the bottom 311b is configured for resealing and liquid retention during operation. The cap 311a may be peeled away to insert the test sample collected by the corer 200 and then reclose it after the assay is completed.

Fig. 3F is a plan view (left view) of the cup housing body 320 when the upper housing 320a and the outer housing 320b are assembled together. The upper housing 320a may include one or more chambers operatively connected. In one embodiment, homogenization chamber 321, filtration chamber 322, and waste chamber 323 are included in housing 320a (left figure). Two breather filters 314 are also added to the upper housing 320 a. The bottom of the assembled cup 320 includes an opening 331a that is connected to the reaction/detection chamber 331 through the inlet and outlet 336 for fluid flow (right drawing). In this embodiment, the reaction/detection chamber 331 is formed when the bottom cover 337 is assembled with the body portion (see fig. 3C). The rotor 340 and rotary valve 350 can be assembled into a cup to form an analysis cartridge (right).

Fig. 3G also shows an external interface at the bottom of the upper housing 320a (top view) and an internal interface at the bottom of the outer housing 320b (bottom view). The two energy director faces 361 (face 1) and 362 (face 2) at the exterior interface of the upper housing 320a interact with the two welded mating faces, faces 363 (face 1) and 364 (face 2) at the interior interface of the bottom of the outer housing 320b, to hold the housing portions 320a and 320b together to form the cup 320. A fluid path 370 is also included to allow liquid to flow to the cup bottom 330. The rotor 340 and the rotary valve 350 are assembled into the cup through the rotor port 340a and the rotary valve port 350a, respectively.

Fig. 3H further illustrates the cup bottom cover 337 of the cup bottom 330 of the cup 300 shown in fig. 3A and 3C. The reaction/detection chamber 331 includes a dedicated sensor area 332 in which a detection sensor (i.e., a glass chip 333) is placed through a glass gasket 334. A glass gasket 334 may be included to seal the glass chip 333 in place to the bottom of the reaction chamber 331 and prevent fluid leakage. Alternatively, adhesives or ultrasonic bonding may be used to fit the layers together. In some aspects of the present disclosure, the glass chip 333 may be configured directly at the bottom of the reaction chamber 331 (e.g., the bottom surface of the sensor region 332) as a component of the cup bottom cover 337, and may be integrated into the cup as a whole. The entire unit may have PMMA (poly (methyl methacrylate)) (also known as acrylic or acrylic glass). The transparent PMMA acrylic glass can be used as an optical window for signal detection.

The reaction chamber 331 includes at least one optical window. In one embodiment, chamber 331 includes two optical windows: a primary optical window and a secondary optical window. In some embodiments, the primary optical window serves as an interface for the reaction chamber 331 to the detection device 100, and in particular to the optical system 1030 (shown in fig. 10A, 10B, and 12A-12C) of the detection device 100. A detection sensor (e.g., a glass chip 333) may be located between the optical window and the interface of the optical system. An optional secondary optical window may be located on one side of the reaction chamber 331; the secondary optical window allows detection of background signals. In some aspects of the present disclosure, the secondary optical window may be configured to measure scattered light.

As shown in fig. 3I, the bottom 330 is assembled with the cup 320. From this bottom perspective view, the bottom surface includes a plurality of interfaces for fluid paths (e.g., fluid inlet/outlet 336) and pump (pump) interface 380, as well as the plurality of interfaces that connect the rotor 340 and rotary valve 350 to the detection device 100.

A device may be included in the cup to prevent fluid flow between the compartments of the assembled cup 300. In one embodiment, a dump valve 315 (shown in FIG. 3C) in the cup housing 320a is included to prevent fluid in the homogenization chamber 321 from flowing to a rotary valve 350 configured at the bottom of the cup 300. The dump valve 315 is held in place for shipping, storage, and end of life by rotating the valve 350 (fig. 3C). The rotary valve 350 locks the dump valve 315 over the filter (e.g., filter assembly 325) during transport and prevents fluid flow after the detection assay is completed. The rotary valve 350 may be actuated in several steps to direct the fluid flow to the appropriate chambers. As one non-limiting example, the relative position of the rotary valve 350 during the test is shown in FIG. 8B.

The rotary valve 350 can be rotated to regulate fluid flow through the chambers inside the cartridge. In some embodiments, the rotary valve 350 may include a valve shaft 351 operatively connected to and locking the dump valve 315 (as shown in fig. 3C) and a valve disc 352 connected to the valve shaft 351 (as shown in fig. 6F, for example). The rotary valve 350 may be attached to the cup by any available means known in the art. In one embodiment, a valve gasket (e.g., gasket 504 shown in FIG. 5B) may be used. Alternatively, the rotary valve may be attached to the cup by a disc spring (e.g., a wave disc spring). In another embodiment, the rotary valve 350 may be secured to the cup by a plurality of compression coil springs (e.g., coil spring 721 shown in FIG. 7J).

In some embodiments, a filter assembly (e.g., the filter 325 shown in fig. 3C, 4A, and 6D) is included in the analysis cartridge. The filter removes large particles and other interfering components, such as fat in the food matrix, from the sample before transferring the processed sample into the reaction chamber 331.

In some embodiments, the filter mechanism may be a filter assembly. The filter assembly may be a simple membrane filter 420. The membrane 420 may be nylon, PE, PET, PES (polyethersulfone), Porex^TMGlass fibers or membrane polymers, such as Mixed Cellulose Esters (MCE), cellulose acetate, PTFE, polycarbonate, PCTE (polycarbonate) or PVDF (polyvinylidene fluoride), and the like. It may be a thin film (e.g. 150 μm thick) with high porosity. In some aspects, the pore size (pore size) of the filtration membrane 420 can be in a range of 0.01 μm to 600 μm, or 0.1 μm to 100 μm, or 0.1 μm to 50 μm, or 1 μm to 20 μm, or 20 μm to 100 μm, or 20 μm to 300 μm, or 100 μm to 600 μm, or any size in between. For example, the pore size may be about 0.02 μm, about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm0 μm, about 4.5 μm, about 5.0 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μma, about 90 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, or about 600 μm.

In some alternative embodiments, the filter assembly may be a complex filter assembly 325 (shown in fig. 4A) that includes multiple layers of filter material. In one example, filter assembly 325 may include a body filter 410 consisting of a coarse filter 411, a depth filter 412, and a membrane filter 420 (fig. 4A). In one embodiment, coarse filter 411 and depth filter 412 may be held by retaining ring 413 to form body filter 410 on membrane filter 420. In other embodiments, the body filter 410 may also include a powder located inside or on top of the filter. The powder may be selected from cellulose, PVPP, resins, etc. In some examples, the powder does not bind nucleic acids and proteins.

In some embodiments, filter assembly 325 may be optimized to remove oil from high fat samples, rather than protein and nucleic acids, to achieve excellent sample cleaning. In other embodiments, the ratio of the depth and width of the filter assembly 325 may be optimized to maximize filtration efficiency.

In some embodiments, the filter assembly 325 can be placed within the filter bed chamber 431 in the disposable cup 320. The bed chamber 431 may be connected to the homogenization chamber 321. A homogenate (homogen) may be supplied to the filter assembly 325 inside the filter bed chamber 431. The filtrate is collected by collection channels 432 (also referred to herein as filtrate chambers) (fig. 4B). The collected filtrate may then exit the fluidic element(s) to flow to the reaction chamber 331 (fig. 3B). In one embodiment, the collected filtrate may be transferred directly from the collection grooves 432 to the reaction chamber 331. In another example, the filtrate may be delivered to the filtrate collection chamber 433 prior to being delivered to the reaction chamber 331 through the inlet/outlet 336 (fig. 3H). Fluid may be delivered to the reaction chamber 331 through a fluid path 370 at the bottom of the cup 320 (as shown in fig. 3G).

In some embodiments, the filtrate collection chamber 433 may further include a filtrate concentrator configured to concentrate the sample filtrate before the sample filtrate flows to the reaction chamber 331 for signal detection. The concentrator may be hemispherical in shape, or a conical concentrator or tall tube.

Thus, the processed sample (e.g., homogenate from chamber 321) is filtered sequentially through coarse filter 411, depth filter 412, and membrane filter 420. The coarse filter 411 may filter large particle suspensions from the sample, e.g. particles larger than 1mm and/or some dyes. The depth filter 412 may remove small particle collections and oil components from a sample (e.g., a food sample). The pore size of the depth filter 412 may range from about 1 μm to about 500 μm, or from about 1 μm to about 100 μm, or from about 1 μm to about 50 μm, or from about 1 μm to about 20 μm, or from about 4 μm to about 15 μm. For example, the pore size of the depth filter 412 may be about 2 μm, or about 3 μm, or about 4 μm, or about 5 μm, or about 6 μm, or about 7 μm, or about 8 μm, or about 9 μm, or about 10 μm, or about 11 μm, or about 12 μm, or about 13 μm, or about 14 μm, or about 15 μm, or about 16 μm, or about 17 μm, or about 18 μm, or about 19 μm, or about 20 μm, or about 25 μm, or about 30 μm, or about 35 μm, or about 40 μm, or about 45 μm, or about 50 μm.

Depth filter 412 may be constructed of, for example, cotton (cotton), including but not limited to raw and bleached cotton, polyester mesh (monofilament polyester fiber), and sand (silica). In some embodiments, the filter material may be hydrophobic, hydrophilic, or oleophobic. In some examples, the material does not bind nucleic acids and proteins. In one embodiment, the depth filter is a cotton depth filter. The size of the cotton depth filter may vary. For example, the aspect ratio of the cotton depth filter may be in the range of about 1:5 to about 1: 20. The cotton depth filter 412 may be configured to correlate total filter volume with the amount of food being filtered.

The membrane filter 420 can remove small particles less than 10 μm in size, or less than 5 μm, or less than 1 μm in size. The pore size of the membrane may be in the range of about 0.001 μm to about 20 μm, or 0.01 μm to about 10 μm. Preferably, the filter membrane may have a pore size of about 0.001 μm, or about 0.01, or about 0.015 μm, or about 0.02 μm, or about 0.025 μm, or about 0.03 μm, or about 0.035 μm, or about 0.04 μm, or about 0.045 μm, or about 0.05 μm, or about 0.055 μm, or about 0.06 μm, or about 0.065 μm, or about 0.07 μm, or about 0.075 μm, or about 0.08 μm, or about 0.085 μm, or about 0.09 μm, or about 0.095 μm, or about 0.1 μm, or about 0.15 μm, or about 0.2 μm, or about 0.25 μm, or about 0.3 μm, or about 0.35 μm, or about 0.15 μm, or about 0.2 μm, or about 0.55 μm, or about 0.5 μm, or about 0.2 μm, or about 0.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm, or about 6.0 μm, or about 7.0 μm, or about 8.0 μm, or about 9.0 μm, or about 10 μm. As described above, the membrane may be a nylon membrane, PE, PET, PEs (polyethersulfone) membrane, glass fiber membrane, polymer membrane such as Mixed Cellulose Ester (MCE) membrane, cellulose acetate membrane, nitrocellulose membrane, PTFE membrane, polycarbonate membrane, track etched polycarbonate membrane, PCTE (polycarbonate) membrane, polypropylene membrane, PVDF (polyvinylidene fluoride) membrane, or nylon and polyamide membrane.

In one embodiment, the membrane filter is a PET membrane filter with a l μm pore size. The small pore size prevents particles larger than l μm from passing into the reaction chamber. In another embodiment, the filter assembly may comprise a cotton filter combined with a 1 μm pore size PET mesh.

In other embodiments, the filter components may be assembled together by any method known in the art, such as by heat welding, ultrasonic welding, or similar processes, which ensures that the assembled material may be die-cut and wrapped without damaging or inhibiting the performance of each filter (either individually or as an integrated filter assembly). In other embodiments, packaging of each portion of the filter assembly enables high speed automated systems to be implemented on a manufacturing assembly line (e.g., a robotic assembly line).

In some embodiments, the filtration mechanism has low protein binding, low nucleic acid binding, or no nucleic acid binding. The filter can be used as a bulk filter to remove fat and emulsifiers as well as large particles, resulting in a filtrate with a viscosity comparable to that of the buffer.

In some embodiments, filter assembly 325 comprising coarse filter 411, depth filter 412, and membrane filter 420 can allow for maximum recovery of Signal Polynucleotide (SPN) and other detection agents.

In other embodiments, the filter assembly 325 may be configured to include a filter 624 (e.g., a mesh filter) inserted into a filter gasket 623, a body filter 622 composed of a coarse filter and a depth filter, and a filter cover 621 (as shown in fig. 6D). In an alternative embodiment, such as in the homogenization chamber 321, a filter gasket 623 may be molded into the bowl as an over-molded part of the bowl 320 (as shown in figures 7E and 7F). The filter 624, body filter 622, and filter cover 621 are inserted into the overmolded gasket to form the functional filter assembly 325.

In some embodiments, the filtration mechanism may complete the filtration process in less than 1 minute, preferably in about 30 seconds. In one example, the filter mechanism is capable of collecting samples in 35 seconds, or 30 seconds, or 25 seconds, or 20 seconds at a pressure of less than 10 psi. In some embodiments, the pressure may be less than 9pis, or less than 8psi, or less than 7psi, or less than 6psi, or less than 5 psi.

In some alternative embodiments, the filtration chamber 322 may include one or more additional chambers, with the implementation chamber being contemplated for filtering the processed sample. As shown in fig. 4B, the filter chamber 322 may also include a separate bed chamber 431 in which a filter assembly 325 (shown in fig. 4A) is inserted and connected to a collection channel 432. The collection channel 432 is configured to collect filtrate that flows through the filter assembly 325, and the channel 432 may be directly connected to a flow cell fluidic element (flow cell fluid) to flow the filtrate to the reaction chamber 331 for signal detection. Optionally, another collection/concentration chamber 433 may be included in the filtration chamber 322, which is configured to collect and/or concentrate the filtrate collected by the collection grooves 432 before delivering the filtrate to the reaction chamber 331 for signal detection. The collection/concentration chamber 433 is collected to the bed chamber 431 through the collection grooves 432.

FIGS. 5A-5C illustrate another embodiment of an analysis cartridge. Fig. 5A shows an alternative assembly of the test cup 300. The various components of the cup 300 of this embodiment are shown in FIG. 5B. According to this embodiment, the cup 300 comprises three parts: a cup top (top of cup) comprising a cup top cover 310; a cup body (body of cup) including a cup receiving groove (cup tank) 320; and a cup bottom (bottom of the cup) comprising a cup bottom cover 330, these parts being operatively connected to form an analysis module. As shown in fig. 5B, the top of the cup is a top cover 310 for sealing the cup in which the test sample is placed for testing. A top gasket 501 may be included to seal the top 310 to the cup 320. The upper cup 320 includes a homogenization chamber, a waste chamber, chambers for wash buffer (e.g., wash 1 chamber (W1), wash 2 chamber (W2)) (as shown in fig. 6B, right view), and an air vent stack (air vent stack) for controlling air and thus fluid flow. A rotor 340 is configured in the homogenization chamber for homogenizing the test sample in the extraction buffer. During assembly, the shape of the rotor may be adjusted to fit the cup. An intermediate gasket 502 is located at the bottom of the upper cup 320 to seal the (cup) body 320 to a manifold 520 having apertures for fluid flow. Manifold 520 is configured to hold filter 325 and fluid path 370 for fluid flow. Another intermediate gasket 503 is added to seal the manifold 520 to the cup bottom 330, where the reaction chamber (e.g., chamber 331), the detection sensor (e.g., glass chip 333), the glass gasket (e.g., gasket 334), and the memory chip (e.g., EPROM) are located. The rotor 340 is sealed to the bottom by an O-ring 505 (shown in fig. 5C). The rotary valve 350 is configured to the cup 300 at the bottom 330 by a valve gasket 504. In another embodiment, the rotary valve 350 may be configured to the cup 300 by spring arms, such as a wave coil spring and a compression coil spring (e.g., coil spring 721 shown in FIG. 7J) located at the cup bottom 330. The configuration of each component of the cup in figure 5A is shown in cross-section in figure 5C.

In accordance with the present disclosure, a third embodiment of a disposable cup 300 is shown in FIG. 6A. Fig. 6B-6G further illustrate the components of the disposable cup 300 in fig. 6A. In this embodiment, the configuration of the detection sensor and the fluid path is further integrated. As shown in fig. 6A, the cartridge includes a top portion 310, a body portion 320, and a bottom portion 330. Rotor 340 is sealed to cup 320 by gasket 612. The rotary valve 350 is assembled to the cassette by a coil spring 613 or alternatively by a compression coil spring (e.g., coil spring 721 shown in fig. 7J) at the cup bottom portion 330. When performing a detection assay, the rotary valve 350 may rotate and move the seal 612 to release (free) the rotor 340, thereby homogenizing the test sample. In this embodiment, a separate panel 631 is provided between the bottom of the bowl 320 and the bottom cover 337, including fluid passages therein. This separate panel 631 with fluid channels is equivalently used as the fluid path 370 of the previous embodiments of the cup (e.g., fig. 3C, 3G, and 3I). The sensor chip 333 may be operatively connected to the sensor region 332 of the reaction chamber 331 and the fluid panel 631 in the bottom cover 337 by a chip PSA 632. In an alternative embodiment, the sensor chip 333 and the fluidic element panel 631 can be combined to form a single thin panel (also referred to as a chip-type channel), thus forming a separate chip-type channel 710 (as shown in FIG. 7A). The chip-based channel 710 will be discussed in detail below.

The cup top 310 may include a top cover 311 (shown in fig. 3E) having two tabs 311a and 311b, and a top cover 312 (shown in fig. 3D). The bowl 320 may be configured to include a plurality of separate chambers including a homogenization chamber 321, a filtration chamber 322, a waste chamber 323, two or more washing spaces (W1 and W2), as shown in fig. 6B (right view). In some examples, the filtration chamber 322 has a vent 611 (shown in fig. 6A). The wetting of the vent 611 may signal the pressure sensor of the electronic device that the chamber 322 is full (fig. 6B). Similar to other designs, at the bottom of the cup 320 (fig. 6B, left) several ports are designed, including port 340a for the rotor 340 and port 350a for the rotary valve 350 (e.g., rotary valve 350 shown in fig. 6F) to assemble a functional cartridge. These ports align with the ports of the bottom cover 337 when the cup bottom cover 337 is sealed to the cup body 320 and the cup is sealed to form an analysis module (e.g., ports 340a and 350a as shown in fig. 6C). The sensor chip 333 is attached to the bottom of the cup 320 by a chip PSA 632 (fig. 6B, left).

Fig. 6C shows a bottom perspective view of the cup bottom cover 337 and the bottom of the cup body 320 aligned with each other, illustrating the position of each component when the test cup is assembled. When the bottom cover 337 and cup 320 are assembled together, a detection chamber is formed with an optical window (331) in which a sensor area 332 holds a sensor chip 333. The optical window of the detection chamber 331 provides a connection to a detector unit (e.g., the detection apparatus 100 in fig. 1 and 9A).

In this embodiment, the fluid panel 631 is located between the bottom of the bowl 320 and the bottom cover 337 (fig. 6A); the fluid panel 631 may be operatively connected to the detection sensor. As one non-limiting example, the fluidic panel 631 is connected to the sensor chip 333 through the chip PSA 632 and provides the necessary fluidic pathways (e.g., 370) for the processed sample to flow to the detection chamber 331 and thus to the sensor chip 333.

In some examples, a filter assembly 325 is inserted into the homogenization chamber 321 to filter the processed sample. In one example, the filter component 325 may be the filter shown in FIG. 4A. In another example, the filter assembly 325 can be configured to include a filter 624 (e.g., a mesh filter), a body filter 622, and a filter cover 621 (fig. 6D), the filter 624 being inserted onto the filter gasket 623. The filter assembly 325 may be secured and controlled by rotating the valve 350 (FIG. 6E). In this embodiment, the filter cover 621 interactively engages the threaded top portion of the rotary valve shaft 351 (FIG. 6E). The rotary valve 350 includes: a valve shaft 351 operatively connected to and locking the filter cover 621; and a valve disc 352 connected to the valve shaft 351 (e.g., in fig. 6F). When the test cup is assembled to the detector unit, the valve disc 352 is connected to the motor of the detector unit.

Fig. 6G shows a bottom perspective view (top view) and a top perspective view (bottom view) of the cup bottom cover 337. The exterior of bottom cover 337 retains the ports (e.g., ports 340a and 350a) and the optical window of sensor area 332 for connection to detection device 100. The inside of the bottom cover 337 includes a coil spring 613 for fixing the rotary valve 350.

In some embodiments, the reaction chamber 331 at the cup bottom cover 337 may include a dedicated sensor region 332 configured to hold a detection sensor for signal detection. In some aspects of the disclosure, the detection sensor may be a solid substrate (e.g., glass surface, chip, and microwell) whose surface is coated with a detection probe, such as a short nucleic acid sequence complementary to SPN that binds to a target allergen. In some examples, the detection sensor held at the sensing region 332 within the reaction chamber 331 may be a glass chip 333 (as shown in fig. 3C and 6A).

In other embodiments, the reaction chamber 331 includes at least one optical window. In one embodiment, the chamber includes two optical windows: a primary optical window and a secondary optical window. Similar to other embodiments, the primary optical window serves as an interface for the reaction chamber 331 to the detection device 100, and in particular to the optical system 1030 (shown in fig. 10A, 10B, and 12A-12C) of the detection device 100. The detection sensor (e.g., the detection region 333' of the chip-based channel 710 and the glass chip 333) may be located between the optical window and the interface of the optical system. An optional secondary optical window may be located on one side of the reaction chamber 331; the secondary optical window allows detection of background signals. In some aspects of the present disclosure, the secondary optical window may be configured for measuring scattered light.

In some embodiments, the detection region 333' of the chip-based channel 710 printed with the nucleic acid molecule (i.e., the DNA chip) and/or the glass chip 333 is aligned with the optical window. In some embodiments, the DNA chip comprises at least one reaction panel and at least one control panel. In some aspects, the reaction panel of the chip faces the reaction chamber 331, which is sandwiched (e.g., shown in fig. 3H and 3I) by the inlet and outlet channels 336 of the cartridge 300. In some embodiments, the reaction panel of the glass chip 333 may be coated/printed with detection probes (e.g., short nucleic acid probes) that hybridize to SPNs with high specificity and binding affinity (binding affinity) for the allergen of interest. The SPNs can then be anchored to the chip when hybridized to the nucleic acid probes.

In a preferred embodiment, the sensor DNA chip (e.g., the chip 333 in fig. 3C, 5B, and 6A, and the detection region 333' in fig. 7B) may include: a reaction panel printed with detection probes comprising short complementary sequences that hybridize to SPNs against the allergen of interest; and two or more control regions (control panels) covalently linked to nucleic acid molecules (as control probes) that are not reactive with SPNs or allergens. When SPN is not bound to the target allergen protein, the complementary probe sequence can only bind to SPN. In some embodiments, the nucleic acid molecules printed on the control panel are labeled with probes (e.g., fluorophores). These control panels provide an optical set-up with a mechanism to normalize the signal output relative to the reaction panel and confirm the functional operating program. An exemplary configuration of the chip 333 or the detection region 333' is shown in fig. 13A.

In another embodiment, the sensor DNA chip (e.g., the chip 333 in fig. 3C, 5B, and 6A, and the detection region 333' in fig. 7B) may include: a reaction panel printed with detection probes comprising short complementary sequences that hybridize to SPNs directed against the allergen of interest; a control region (control panel) covalently linked to a control nucleic acid molecule; and one or more fiducial points that can guide image processing and provide a self-correcting mechanism for an image detector (e.g., the camera detector in fig. 15A). An exemplary configuration of the chip 333 or the detection region 333' is shown in fig. 13C.

In some embodiments, the DNA coated chip can be prepackaged into the reaction chamber 331 of the cartridge, for example at the sensing region 332. In other embodiments, the DNA coated chip can be packaged separately from a disposable cartridge (e.g., cup 300 in fig. 1). In other embodiments, the DNA chip 333 may be attached to the fluidic panel 631 shown in fig. 6A. In other embodiments, the DNA chip may be integrated into a chip-based channel as a dedicated detection region for the chip-based channel (e.g., detection region 333' of chip-based channel 710 shown in FIG. 7B).

Another alternative embodiment of an analysis cartridge is provided in the present disclosure. The configuration of the test cup of this alternative embodiment is shown in fig. 7A, where test cup 300 comprises a similar configuration of compartments (e.g., as shown in fig. 6A) comprising: a cup top 310; a cup 320 configured to include a homogenization chamber, a filtrate chamber, a plurality of washing chambers, and a waste chamber; and a cup bottom 330. This design is simple and requires few parts. In this embodiment, chip-based channels 710 combining fluidic panel 631, chip 333, and chip PSA 632 into a single sheet are provided to replace these components. Chip-style channel 710 may be connected to cup 320 (fig. 7C) via port connection 711 through gasket 701 (fig. 7A) and bottom cover 337. Alternatively, chip-style channel 710 may be soldered to the cup through sealing face 712 (e.g., in an alternative embodiment shown in fig. 7D).

In some embodiments, chip-based channel 710 includes a fluid path and a sensor chip having detection probes immobilized thereon, the sensor chip being made of a single thin plastic polymer. According to the present disclosure, the chip-based channel 710 may be a piece of plastic, with certain regions (FIG. 7B) configured as detection regions 333' (i.e., equivalent to separate DNA chips 333 in other embodiments). The chip-based channel 710 can include a fluid channel (e.g., pathway 370 in fig. 7B) connected to the detection region 333'. The detection zone 333 'may be sandwiched by the inlet and outlet channels 336' (fig. 7B). The chip-type channel 710 may be made of optically transparent resin (e.g., COC, COP, and PMMA).

In some embodiments, nucleic acid-based detection probes are printed on the detection zones 333' of the chip-based channels 710 by UV radiation. In some examples, the detection zone 333' further includes a control probe immobilized thereon. The detection probes and control probes are immobilized to form separate reaction and control panels. In some embodiments, the nucleic acid probes and control probes are printed on the detection regions 333' of the chip-based channels 710, as shown in FIG. 13C. The detection probes and the control probes are printed on the reaction panel 1312 and the control panel 1313, respectively. Within each panel, the detection probes and control probes are printed in a checkerboard (checkerboard) pattern, such as the pattern shown in FIG. 13D.

Fig. 7C and 7D show perspective views of chip-based channel 710. In one embodiment, chip-style channel 710 is retained by port connector 711 (FIG. 7C). A vacuum (e.g., of detection device 100) is connected to chip-based channel 710 through port connector 711. In another embodiment, the chip-style channel 710 is sealed to the cup bottom 337 via a face seal 712 (FIG. 7D). The overmolding of the chip-style channels 710 and cup bottoms 330 will result in a seamless bonding of the components. The components may be overmolded as a single component using any overmolding and casting technique, such as an injection molding process.

In some embodiments, the solid substrate (e.g., chip-type channel 710) on which the detection probes are immobilized can be a glass with high optical transparency, such as borosilicate glass and soda glass (soda glass).

In other embodiments, the solid substrate (e.g., chip-based channel 710) on which the detection probes are immobilized can be made of a plastic material with high optical transparency. As one non-limiting example, the substrate may be selected from the group consisting of: polydimethylsiloxane (PDMS), Cyclic Olefin Copolymer (COC), Polymethylmethacrylate (PMMA), Polycarbonate (PC), Cyclic Olefin Polymer (COP), Polyamide (PA), Polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), Polystyrene (PS), Polyoxymethylene (POM), Polyetheretherketone (PEEK), Polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polyvinyl alcohol, polyacrylate, polybutylene terephthalate (PBT), Fluorinated Ethylene Propylene (FEP), perfluoroalkoxy alkane (PFA), polypropylene carbonate (PPC), Polyethersulfone (PEs), polyethylene terephthalate (PET), cellulose, poly (4-vinylphenyl chloride) (PVBC),

Hydrogels, Polyimides (PI), 1, 2-Polybutadiene (PB), fluoropolymers-and copolymers (e.g., poly (tetrafluoroethylene) (PTFE), perfluoroethylene propylene copolymer (FEP), Ethylene Tetrafluoroethylene (ETFE)), norbornene group-containing polymers, polymethylmethacrylate, acrylic polymers or copolymers, polystyrene, substituted polystyrenes, polyimides, silicone elastomers, fluoropolymers, polyolefins, epoxies, polyurethanes, polyesters, polyethylene terephthalate, polyperfluorones, and polyetherketones, and combinations thereof. The chip and chip-like channels may be prepared by injection molding.

In another embodiment of the test cup 300 shown in FIG. 7E, the cup is further optimized for improved performance and for manufacturing. In this embodiment, a filter gasket 623 is overmolded to the interior of the bowl, for example in the homogenization chamber 321 (fig. 7F). Fig. 7H shows a cross-sectional view of the over-molded seal 713, which combines the components into a single component. Overmolding facilitates the manufacturing process to form the individual pieces. In this embodiment, the top of the valve shaft 351 of the rotary valve 350 includes a cam 353 (fig. 7G) that interacts with the filter cover 621 to provide rotational motion (fig. 7F, right). Fig. 7I shows a bottom perspective view (bottom view) of the cup bottom 337 (top view) and the cup 320. In this embodiment, the rotary valve 350 is secured in the test cup body 320 by a plurality of compression coil springs 721 at the cup bottom cover 337 (fig. 7J). Fig. 7J also shows a compression coil spring 721 at the cup bottom 337. Four coil springs 721 may be located at each corner (horns) of the rotary valve port 350a to secure the valve 350. In this embodiment, chip-style channel 710 may be soldered to the bottom of cup 320. For example, chip-style channel 710 may be laser welded to the bottom of cup 320. In one example, fig. 7K shows a bead material 722 at the bottom of the cup 320 for laser welding.

The cup bottom 330 is configured to close the disposable test cup 300 and provide a means for coupling the test cup 300 to the test device 100 in the various embodiments discussed herein. In some embodiments, the bottom side of the bottom component 330 of the cup 300 shown in fig. 3H includes a plurality of interfaces for connecting the cup 300 to the detection apparatus 100 for operation, including: a homogenizing rotor interface 340a that may couple the homogenizing rotor 340 to a motor in the apparatus 100 for controlling homogenization; a valve interface 350a that can couple the rotary valve 350 to a motor in the device 100 for controlling valve rotation; and a pump interface 380 for connecting a pump in the test device 100.

In some embodiments, a valve system is provided to control fluid flow of sample, detection agents, buffers and other reagents, and waste through different portions of the cartridge (e.g., individual chambers within the cup). In addition to the flexible membrane, foil seal, and pinch valve discussed herein, other valves may be included to control fluid flow during the detection assay, including swing check valves, gate valves, ball valves, rotary valves, custom valves, or other commercially available valves. For example, a gland seal (gland seal) or rotary valve 350 may be used to control the flow of the processed sample solution within the cup 300. In some examples, pinch valves or rotary valves are used to completely isolate the fluid from other internal valve components. In other examples, an air operated valve (e.g., an air operated pinch valve) is used to control fluid flow, which is operated by a pressurized air supply.

In one embodiment, the means for controlling fluid flow within the cup chamber can be included in, for example, the cup bottom assembly 330 and/or the cup 320. The device may include a flow channel, a conduit, a valve, a gasket, a vent, and an air connection. In other embodiments, the means for fluid flow may be configured as a separate component in the cup, such as the fluid panel 631 shown in fig. 6A.

In other embodiments, the valve system of the present disclosure may include an additional vent included in the test cup 300 to control air flow when a DNA-coated glass chip is used as a detection sensor. During the allergen detection assay, the DNA chip may be flushed (purge) with air. Individual air inlets may be opened according to the requirements of the system. A valve system as discussed herein may be used to keep the vent unit inactive until the time of use. One or more air ports allow air to enter the cartridge (e.g., cup 300) and one or more vents allow air to enter the various chambers when fluid is added to or removed from the chambers. These vents may also have a membrane contained therein to prevent spillage (spill) and act as a mechanism to control the amount of fluid filling by occluding the venting membrane, stopping further flow and filling functions.

In a preferred embodiment, a rotary valve 350 (shown in FIGS. 3C, 5B, 6A and 6F, and 7A) may be used to control and regulate fluid flow and flow rate in test cup 300. The rotary valve 350 includes a valve shaft 351 and a valve disc 352 (fig. 6F and 7G), which may be operated by an associated detection device (e.g., device 100). In some embodiments, the rotary valve 350 may be positioned at a particular angle by rotating the valve member in a counter-clockwise (CCW) or Clockwise (CW) direction at each step of a repeated wash and air flush cycle during the course of testing an assay. The air holes may allow air to enter. Air is drawn through the system via vacuum pressure to perform an air flush function. The angle may be in the range of about 2 ° to about 75 °.

As one non-limiting example, the valve may be about 38.5 ° relative to the gas vent, with the pump 1040 closed and the reaction chamber 331 dry (referred to as a home position). After processing and homogenization of the test sample, the pump is opened and the valve 350 is CCW (counterclockwise) rotated and stopped at an angle of about 68.5 °, allowing the processed sample to be delivered to the filtration chamber 322. Next, the valve member may be rotated again in a different direction while stopping at a different angle, for example, at about 57 deg. to allow the washing buffer to flow to the reaction chamber 331, and at about 72 deg. to flush the DNA chip with air. After pre-washing the DNA chip, the valve member can be rotated to a home position at about 38.5 deg.. The processed sample solution is pulled through the filter assembly 325. After filtration, the valve member may be rotated and stopped at an angle of about 2 °, allowing the collected filtrate to flow into the reaction chamber 331 where the chemical reaction takes place. The valve 350 will rotate and stop at about 57 deg. to flow the washing buffer to the reaction chamber 331 and at about 72 deg. to flush the DNA chip with air. The washing and air rinsing steps may be repeated one or more times until the optical measurements show a clean background.

In other embodiments, the rotary valve 350 is operatively connected to a filter cap 621 (fig. 6E). For example, during shipping of the test cup 300, the filter cap may lock the rotary valve 350.

In one embodiment, the valve system may be a rotary valve as shown in fig. 8A and 8B. In this embodiment, the rotary valve 350 is positioned to control air ingress and fluid flow. Such positioning may drive homogenization in the homogenization chamber 321, filtration and collection of filtrate (F), sample washing (e.g., wash 1(W1) and wash 2(W2)), and waste collection (shown in fig. 8A). In step 1 of fig. 8B, the rotary valve 350 is in a closed position, wherein no connection is established between any one of the chambers. In step 2 of fig. 8B, the rotary valve 350 connects the wash 1 chamber W1 to the reaction chamber 331 to flush the reaction chamber 331 and then push the wash buffer out to the waste chamber 323. In step 3 of fig. 8B, the rotary valve 350 connects the homogenization chamber 321 to the filtrate chamber F to effect the filtration step. In step 4 of fig. 8B, rotary valve 350 connects filtrate chamber F to reaction chamber 331 to send the filtrate to reaction chamber 331 for reaction and analysis. In step 5 of fig. 8B, the rotary valve 350 connects the wash 2 chamber W2 to the reaction chamber to flush the reaction chamber 331 again.

In some embodiments, the extraction buffer may be pre-stored in an analysis cartridge, e.g., homogenization chamber 321 of cup 320, e.g., in a foil-sealed reservoir, like food processing reservoir 801 (fig. 8C). Alternatively, the extraction buffer may be stored separately in a separate buffer reservoir in the bowl 320, a reservoir similar to the wash buffer storage reservoir 802 (in the buffer storage chamber 324 (optional), as shown in fig. 8C). The extraction buffer and wash waste after sample homogenization may be stored in separate waste reservoirs 803 in the waste chamber 323. The waste chamber 323 has sufficient volume to store a volume greater than the amount of fluid used during the detection assay.

According to the present disclosure, the homogenizing rotor 340 may be configured small enough to fit into a disposable test cup 300, in particular into the homogenizing chamber 321, where the homogenizer processes the sample to be tested. Furthermore, the homogenizing rotor 340 may be optimized to increase the efficiency of sample homogenization and protein extraction. In one embodiment, the homogenizing rotor 340 may comprise one or more blades or their equivalents at the proximal end. In some examples, the rotor 340 may include one, two, three, or more blades. The homogenizing rotor 340 is configured to pull the test sample from the food corer 200 into the bottom of the homogenizing chamber 321.

Alternatively, the homogenizing rotor 340 may also include a central rod through the rotor that connects to the second port nozzle through the cup 320. The central rod may serve as an additional bearing surface, or for transferring rotational motion to the rotor 340. When the rotor 340 is mounted to the cup through a port in the bottom of the cup (e.g., port 340a), the blade tips may remain submerged within the extraction buffer during operation. In another alternative embodiment, the homogenizing rotor 340 may have an extension to provide a channel (pass) through the bottom of the cup; the channel may serve as a second bearing support and/or an additional location for power transfer. In this embodiment, the lower portion of the rotor is tapered to fit to the shaft, forming a one-piece rotor. According to the present disclosure, the depth of the blades of the homogenizing rotor 340 with or without a central rod is configured to ensure that the blade tips are in the fluid during sample processing.

In contrast to other homogenizers (e.g., U.S. patent No. 6,398,402; incorporated herein by reference in its entirety), the custom vane core of the present disclosure rotates and pulls and forces food into the toothed surface of the custom shroud. The homogenizer rotor may be made of any thermoplastic material, including but not limited to Polyamide (PA), Acrylonitrile Butadiene Styrene (ABS), Polycarbonate (PC), High Impact Polystyrene (HIPS), and acetal (POM).

The disposable cartridge can be any shape, such as circular, elliptical, rectangular, or oval. Any of these shapes may be provided with finger-like cuts or notches. The disposable cartridge may be asymmetric or symmetric.

Optionally, a label or foil seal may be included on the top of the cap 311 to provide a final fluid seal and identification of the test cup 300. For example, the indication of peanuts shows that the disposable test cup 300 is used to detect peanut allergens in a food sample.

Detection device

In some embodiments, the detection device 100 may be configured with: an outer housing 101 providing a support surface for the components of the detection device 100; and a cover 103 that opens the test device 100 for insertion of the disposable test cup 300 and covers the cup during operation. The small cover may be located on one side of the device (as shown in fig. 1 and 9A) or in the center (not shown). In some aspects of the present disclosure, the cover may be transparent, allowing all operations to be visible through the cover 103. The device may also include a USB port 105 for transferring data.

One embodiment of an allergen detection device 100 according to the present disclosure is shown in fig. 1 and 9A. As shown in fig. 1, the test device 100 includes an outer housing 101 that provides support for holding the various components of the test device 100 together. The outer housing 101 may be formed of plastic or other suitable supporting material. In other embodiments, the device may be made of aluminum. The device also has a port or receptacle 102 for docking with a test cup 300 (fig. 1 and 9A).

In order to perform an allergen detection test, the detection apparatus 100 is provided with: means for handling the homogenizing assembly (e.g. a motor) and the necessary connectors connecting the motor to the homogenizing assembly; means to control the rotary valve (e.g., a motor); means for driving and controlling the flow of the treated sample solution during the allergen detection test; an optical system; means for detecting a fluorescent signal from a detection reaction between the allergen in the test sample and the detection agent; means for visualizing the detected signals, including converting and digitizing the detected signals; a user interface for displaying the test results; and a power source.

Viewed from the transparent cover 103 (fig. 9A), the device 100 has an interface comprising an area for coupling components of the cartridge 300 (when inserted) for operating the detection reaction (fig. 9B). These areas include: a homogenizing nozzle 910 for coupling the rotor 340 to the motor; a vacuum nozzle 920 for coupling the cup with a vacuum pump; a rotary valve drive mouthpiece 930 for coupling the rotary valve 350 to a valve motor; and a protective glass 940 aligned with the sensor region 333' of the chip-based channel 710 or the glass chip 333 through the optical window of the reaction chamber 331. A data chip reader 950 is also included to read the data chip 335. The pin 960 is used to aid in the placement of the cup 300 in the receptacle of the device 100.

In one embodiment of the present disclosure, as shown in figure 10A, the components of the inspection apparatus 100 are integrated for all movement and actuation for operating the inspection reaction, including the motor 1010, which may be connected to the homogenizing rotor 340 within the homogenizing chamber 321 in the cup 320. The electric machine 1010 may be connected by a multi-component coupling assembly comprising: a gear train/drive platen (cylinder) for driving the rotor during homogenization in the allergen detection test; a valve motor 1020 for driving the rotary valve 350; an optical system 1030 connected to the reaction chamber 331 (not shown) or the chip-based channel 710 within the disposable test cup 300; a vacuum pump 1040 for controlling and regulating air and fluid flow (not shown in fig. 10A); a PCB display 1050; and a power supply 1060 (in fig. 10B). A means for holding a test cup, i.e., a cup holder (cup retention)1070, is included for holding the test cup 300. Each of which will be described in detail below.

1. Homogenizing assembly

In one embodiment, the motor 1010 may be connected to the homogenizing rotor 340 inside the test cup 300 by a multi-part rotor coupling assembly. The rotor coupling assembly may include: a coupling directly coupled to the distal shroud of the rotor 340; and a gear head that is part of a gear train or drive (not shown) for connection to the motor 1010. In some embodiments, the coupling may have different dimensions at each end thereof, or the same dimensions at each end of the coupling. The distal end of the coupling assembly may be connected to the rotor 340 through a rotor port 340a at the cup bottom 330. Other alternative means for connecting the motor to the homogenizing rotor 340 may also be used to form a functional homogenizing assembly within the scope of the present disclosure.

In some embodiments, the motor 1010 can be a commercially available motor, for example, a Maxon motor system: maxon RE-max and/or Maxon A-max (Maxon Motor ag, San Mateo, Calif., USA).

Optionally, a heating system (e.g., resistive heating or peltier heater) may be provided to increase the temperature of homogenization, thereby increasing the effectiveness of sample dissociation and shortening the processing time. The temperature may be raised to 60 ℃ to 95 ℃ but below 95 ℃. The elevated temperature may also facilitate binding between the detector molecule and the allergen being detected. Optionally, a fan or peltier cooler may be provided to rapidly reduce the temperature after the test is conducted.

The motor 1010 drives the homogenization assembly to homogenize the test sample in the extraction buffer and dissociate/extract the allergen proteins. The processed sample solution may be pumped or pressed through the flow tube to the next chamber for analysis, e.g., into the reaction chamber 331 where it is mixed with pre-loaded detection molecules (e.g., aptamer magnetic bead conjugates) for detection testing. Alternatively, the processed sample solution may be pumped or pressed through the flow tube to the filter assembly 325 and then to the filtrate chamber 322 before being passed to the reaction chamber 331 for analysis.

2. Filtration

In some embodiments, a means for controlling the filtration of the processed test sample may be included in the detection device. The food sample will be pressed through a filter membrane or filter assembly before the extraction solution is delivered to the reaction chamber 331 and/or other chambers for further processing (e.g., washing). One example is one or more filter membranes. The membrane provides for the filtration of specific particles from the treated protein solution. For example, the filtration membrane may filter particles up to about 0.1 μm to about 1000 μm, or about 1 μm to about 600 μm, or about 1 μm to about 100 μm, or about 1 μm to about 20 μm. In some examples, the filtration membrane can remove particles up to about 20 μm, or about 19 μm, or about 18 μm, or about 17 μm, or about 16 μm, or about 15 μm, or about 14 μm, or about 13 μm, or about 12 μm, or about 11 μm, or about 10 μm, or about 9 μm, or about 8 μm, or about 7 μm, or about 6 μm, or about 5 μm, or about 4 μm, or about 3 μm, or about 2 μm, or about 1 μm, or about 0.5 μm, or about 0.1 μm. In one example, the filter membrane can remove up to about 1 μm of particles from the processed sample. In some aspects, filter membranes may be used in combination to filter out specific particles from an assay for analysis. The filtration membrane may comprise a multi-stage filter. The filter membrane and/or filter assembly may be of any configuration relative to a flow valve. For example, the flow valve may be above, below, or between any stages of filtration.

In some embodiments, the filter assembly may be a complex filter assembly 325 as shown in fig. 4A, wherein the processed sample is filtered sequentially through a coarse filter 411, a depth filter 412, and a membrane filter 420. In other embodiments, the filter assembly 325 may be a filter stack as shown in fig. 6D.

3. Pump and fluid movement

According to the present disclosure, a device for driving and controlling the flow of a processed sample solution is provided. In some embodiments, the device may be a vacuum system or an external pressure. As one non-limiting example, the device may be a platen configured for multiple functions (e.g., welded plastic clamshell) because it may support the axis of the gear train and may provide for pumping (sealed air channel) of the vacuum applied from the pump 1040 to the test cup 300. Pump 1040 can be connected to test cup 300 through pump port 920 located at the bottom (FIG. 9B), which connects to pump interface 380 on bottom 330 of test cup 300 (FIG. 3G) when the cup is inserted into the device.

Such as a piezoelectric micropump (e.g., Takasago Electric corporation, republic of japan) or a peristaltic pump (flow) may be used to control and automatically adjust the flow to a target flow rate. The flow rate of the pump can be adjusted by changing the driver voltage or driving frequency. As one non-limiting example, the pump 1040 may be a peristaltic pump. In another embodiment, pump 1040 may be a piezoelectric pump currently on the market with specifications indicating the aliquoting function (aliquot function) that it may be suitable for required to flow the filtered sample solution into different chambers within test cup 300. The pump 1040 may be a vacuum pump or another small pump configured for laboratory use, such as a KBF pump (KNF Neuberger, Trenton, NJ, USA).

Alternatively, a syringe pump, a septum, and/or a micro-peristaltic pump may be used to control fluid movement during detection of the assay and/or support fluid elements. In one example, an air operated diaphragm pump may be used.

4. Rotary valve control

In some embodiments, the rotary valve 350 (e.g., as shown in FIG. 6F) used to control the fluid flow needs to be in a precise position. To this end, a device is provided for controlling a rotary valve, and a control mechanism is capable of rotating the valve in both directions and stopping precisely at a desired position. In some embodiments, the device 100 includes a valve motor 1020 (shown in fig. 10A). As shown in fig. 11A, the valve motor 1020 may be a low cost, dc gear motor 1110 with two low cost optical sensors (sensors 1131 and 1132), and a microcontroller. The output coupling 1120 interfaces with the rotary valve 350. In some embodiments, the output coupling 1120 has a "half-moon" shelf (shelf)1170, as shown in fig. 11B, that outputs the optical sensor 1131 with a protruding half (protruding half) break (interrupt). The output optical sensor signal is switched between high and low (toggle) depending on whether the protruding bracket interrupts the sensor. A Microcontroller (MCU) detects these transitions and derives the absolute position of the output from the signal. The location of these transitions is important and application specific, as these transitions are used to account for gear backlash during direction changes.

A direct drive motor shaft (direct motor shaft)1140 has a paddle wheel (paddle) that interrupts the direct drive shaft optical sensor 1132, allowing the direct drive shaft optical sensor 1132 to output a series of pulses, the number of pulses per revolution being determined by the number of paddle wheels on the wheel 1150. The MCU reads the series of pulses and determines the degree shift (degrees moment) of the output link. The resolution depends on the number of paddle wheels of the direct drive shaft encoder wheel 1150 and the gear reduction ratio of the gear box 1160.

As long as the position of the output link transitions, the number of paddle wheels on the direct drive encoder wheel 1120, and the gear ratio are known, the MCU interrupts the outputs of the two optical sensors and can drive the outputs to the desired positions. During a change of direction, the motor must rotate a fixed amount selected to overcome the backlash of the gears before the output transition is seen. Once this fixed amount is overcome, the MCU can start counting the drive signal pulses at the next output signal transition and be sure that these pulses correspond to an accurate output of position and movement.

5. Optical system

The detection device 100 of the present disclosure includes an optical system that detects an optical signal (e.g., a fluorescent signal) generated by an interaction between an allergen in a sample and a detection agent (e.g., an aptamer and SPN). The optical system may comprise different components and variable configurations depending on the type of fluorescence signal to be detected. The optical system is adjacent to and aligned with the detection cartridge, e.g., the primary optical window and optionally the secondary optical window of the reaction chamber 331 of the test cup 300, as described above.

In some embodiments, optical system 1030 can include excitation optics 1210 and emission optics 1220 (fig. 12A and 12B). In one embodiment, as shown in fig. 12A, excitation optics 1210 may include: a Light Emitting Diode (LED)1211 configured to transmit the excitation optical signal to a sensing region (e.g., 332) in the reaction chamber 331; a collimating lens 1212 configured to focus light from the light source; a filter 1213 (e.g., a band pass filter); a focusing lens 1214; and an optional LED power monitor photodiode. The emissive optic 1220 may include: a focusing lens 1221 configured to focus at least a portion of the allergen-dependent optical signal onto a detector (photodiode); two filters, including long pass filter 1222 and band pass filter 1223; a converging lens 1224 configured to collect light emitted from the reaction chamber and an aperture (aperture) 1225. The emissive optics collect light emitted from the solid surface (e.g., DNA chip 333) in the detection chamber 331 and this signal is detected by a detector 1230 configured to detect the allergen-dependent optical signal emitted from the sensing region 332. In some aspects, the excitation power monitor may be integrated into the LED (not shown in fig. 12A).

The light source 1211 is arranged to transmit excitation light that excites in a wavelength range. Suitable light sources include, but are not limited to, lasers, semiconductor lasers, Light Emitting Diodes (LEDs), and organic LEDs.

An optical lens 1212 may be used with the light source 1211 to provide the excitation source light to the fluorophores. Optical lens 1214 may be used to limit the range of excitation light wavelengths. In some aspects, the filter may be a band pass filter.

The fluorophore-labeled SPN for the target allergen is capable of emitting an optical signal (e.g., fluorescence) in response to excitation light in at least one excitation wavelength range dependent on allergen binding in at least one emission wavelength range.

In some embodiments, the emission optics 1220 are operable to collect these emissions (emissions) upon interaction between the detection agent from the reaction chamber 331 and the target allergen in the test sample. Optionally, a mirror may be interposed between the emissive optic 1220 and the detector 1230. The mirror can be rotated over a range of angles (e.g. from 1 deg. to 90 deg.), which can help to form a compact optical unit inside a small portable detection device.

In some embodiments, more than one emission optical system 1220 may be included in the detection device. As one non-limiting example, three photodiode optical systems may be provided to measure the fluorescence signals from the unknown test area and two control areas on the glass chip (see, e.g., fig. 13B). In other aspects, additional converging lenses 1224 may also be included in emissive optics 1220. The converging lens may be configured to detect several different signals from the chip 333. For example, when a detection assay is carried out using a DNA glass chip, more than two control regions may be constructed on a solid surface in addition to the detection region for allergen detection. When measuring allergen-derived signals, the internal control signals from each control region can be detected simultaneously. In this case, more than two converging lenses 1224 may be included in the optical system 1030, one lens 1224 for signals from the detection area and the remaining converging lenses 1224 for signals from the control area.

A detector (e.g., photodiode) 1230 is arranged to detect light emitted from the fluidic chip in the emission wavelength range. Suitable detectors include, but are not limited to, photodiodes, Complementary Metal Oxide Semiconductor (CMOS) detectors, photomultiplier tubes (PMTs), microchannel plate detectors, quantum dot photoconductors, phototransistors, photoresistors, Active Pixel Sensors (APS), gaseous ionization detectors, or Charge Coupled Device (CCD) detectors. In some aspects, a single and/or universal detector may be used.

In some embodiments, the detector 1230 may be an image detector, such as a camera described below.

In some embodiments, the optical system 1030 can be configured to detect a fluorescent signal from a solid substrate sensor (e.g., the DNA chip 333 shown in fig. 13A or the chip-based channel 710 shown in fig. 7A-7C). The DNA chip can be configured to contain a central reaction panel, which is labeled as an "unknown" signal region on the chip (fig. 13A), and at least two control regions at various locations on the chip (fig. 13A). In this case, the optical system 1030 is configured to simultaneously measure the detection signal and the internal control signal (fig. 13B).

In one example, the optical system 1030 includes two converging lenses 1224 and corresponding optical components, such as a control array photodiode for each lens 1224. FIG. 12B shows a side view of the optical system 1030 shown in FIG. 12A inside the detection apparatus 100. In this embodiment, the optical system includes two converging lenses 1224: one for collecting control array signals from the DNA chip (e.g., the two signals 1301 and 1302 shown in fig. 13B) and one for unknown detection signals from the DNA chip (e.g., the detection signal 1302 shown in fig. 13B). In other aspects, the focusing lens 1224 may be configured to collect signals from the detection regions 333' of the chip-based channels 710, such as one signal from the reaction panel 1312 and another signal from the control panel 1313 shown in fig. 13C. For each optical path, one signal array diode 1241 (e.g., LED diode 1211 shown in fig. 12A) and two control assay photodiodes 1242 are included. In addition, two prisms 1243 may be added to two converging lenses (1224) configured to collect signals from two control areas. The prism 1243 may bend the control array light to the photodiode sensor area.

In some embodiments, the optical system 1030 can be configured in a straight pattern as shown in fig. 14A. Excitation optics 1410 is configured to transmit excitation optical signals to glass chip 333 (e.g., a DNA-coated chip) in reaction chamber 331, which may include LED1411, collimating (collimating) lens 1412, band pass filter 1413, and cylindrical lens 1414. The cylindrical lens 1414 may line the excitation light to cover the reaction panel and the control panel on the glass chip (e.g., fig. 13B). The emissive optics 1420 aligned with the glass chip 333 may include a focusing lens 1421 configured to collect light emitted from the glass chip 333, a band pass filter 1422a, a long pass filter 1422b, and a focusing lens 1423 configured to focus at least a portion of the allergen-based optical signal onto the chip reader 1430. The chip reader 1430 is composed of three photodiode lenses 1431, two control array photodiodes 1432, a signal array photodiode 1433, and a collection PCB1434 (fig. 14A). In some embodiments, the converging lens 1421 may be shaped to include a concave (concave) first surface to optimize imaging and minimize stray light (stray light).

As one non-limiting example, excitation optics 1410 and emission optics 1420 can be folded and configured into stepped bore 1480 in apparatus 100 (see fig. 14C). Excitation folding mirror 1440 and converging folding mirror 1450 can be configured to minimize the light paths from excitation optics 1410 and emission optics 1420, respectively (in fig. 14B). The minimized volume allows the laser to be modulated at a frequency that minimizes interference from ambient light sources. A photodiode shield 1460 may be added to cover and protect the chip reader 1430 shown in fig. 14A. The reader 1430 is then positioned adjacent to the converging lens 1421 to minimize stray light. Fig. 14C shows an example of a stepped hole 1480 for holding emissive optic 1420 in the device. An aperture 1470 of the condensing lens 1421 is shown in fig. 14C.

The LED source (e.g., LED1411) can be modulated and/or polarized and oriented to minimize reflection from the glass chip. Thus, the chip readers may be synchronized to measure the modulated light.

Fig. 15A shows another embodiment of an optical system 1030. In this embodiment, the optical system 1030 includes an image detector. The image detector may be a camera 1531 as part of the signal reader 1530. The camera may capture a reaction image of the detection region 333' of the chip-based channel 710 or the sensor DNA chip 333. As one non-limiting example, the optical system 1030 shown in fig. 15A includes: excitation optics 1510 comprising an excitation filter 1513, a collimating lens 1512, and a laser diode 1511; emissive optics 1520 including a converging lens 1521, a bandpass filter 1522a, a long pass filter 1522b (e.g., a colored glass long pass filter), and a focusing lens 1523; and a signal reader 1530 including a camera 1531. Each of the optical systems may be configured in an optical housing, such as optical housing 1540 in fig. 15A, configured to hold components of emissive optics 1520.

Fig. 15B shows a cross-sectional view of the optical system of fig. 15A, which is assembled inside the detection apparatus 100. From this cut-away side view, excitation optics 1510 and emission optics 1520 are assembled into optical housings, respectively. A protective window 1501 may be added to protect the optical components. Optionally, a laser tuning mount 1502 may be included to tune the laser diode 1511 inside the excitation optics 1510. The camera 1531 captures the reaction image and the raw image is collected and processed. The detection result can be displayed through the display PCB 1050.

The optical system 1030 described above is an illustrative example of some embodiments. Alternative embodiments may have different configurations and/or different components.

In other embodiments, a computer or other digital control system may be used to communicate with light filters, fluorescence detectors, absorption detectors, and scatter detectors. A computer or other digital control system controls the optical filter to subsequently illuminate the sample with each of the plurality of wavelengths while measuring the absorption and fluorescence of the sample based on signals received from the fluorescence and absorption detectors.

6. Display device

As shown in the cut-away side view of fig. 10B, a Printed Circuit Board (PCB)1050 is connected to the optical system 1030. The PCB1050 may be configured to be compact and have the size of the test device 100, and at the same time, may provide enough space to display the test results.

Thus, the test results may be displayed with backlit icons, LED or LCD screens, OLEDs, segmented displays, or attached cell phone applications. The user can see an indication that the sample is being processed, that the sample has been completely processed (total protein indicator), and the test results. The user is also able to see the status of the battery and what type of cassette (barcode on cassette or LED assembly) is placed in the device. The test results will be shown as, for example, (1) actual numbers, ppm or mg; or (2) a binary result, yes/no; or (3) risk analysis-high/medium/low or high/low, risk present; or (4) the ppm range, less than 1/1-10 ppm/greater than 10 ppm; or (5) mg, less than 1 mg/between 1mg and 10 mg/greater than 10 mg. The results may also be displayed as numbers, colors, icons, and/or letters.

In accordance with the present disclosure, the detection device 100 may also include other features, such as a device for providing power and a device for providing process control. In some embodiments, one or more switches are provided to connect the motor, micropump and/or gear train or drive to the power source. These switches may be simple micro-switches that can turn the detection means on and off by connecting and disconnecting the battery.

Power supply 1060 may be a lithium ion AA format battery, but may be any commercially available battery suitable for supporting small medical devices, such as a Rhino 610 battery, Tumtigy Nanotech high dischargeable lithium battery, or a Pentax D-L163 battery.

In the description herein, it is to be understood that all enumerated connections between components may be either direct or indirect operative connections. Other components may also include those disclosed in U.S. provisional application 62/461,332 filed on 21/2/2017; the contents of which are incorporated herein by reference in their entirety.

Detection assay

In another aspect of the present disclosure, allergen detection tests are provided that are implemented using the detection assemblies and systems, detection agents, and detection sensors of the present disclosure.

As one non-limiting example, the allergen detection test comprises the steps of: (a) collecting a quantity of a test sample suspected of containing the allergen of interest, (b) homogenizing the sample and extracting the allergen protein using an extraction/homogenization buffer, (c) contacting the treated sample with a detection agent that specifically binds the allergen of interest; (d) contacting the mixture in (c) with a detection sensor comprising a solid substrate printed with nucleic acid probes; (e) measuring a fluorescent signal from the reaction; and (f) processing and digitizing the detected signals and visualizing the interaction between the detection agent and the allergen.

In some aspects of the disclosure, the method further comprises the steps of: unbound compounds are washed from the detection sensor to remove any non-specific binding interactions.

In some aspects of the disclosure, the method further comprises the steps of: the treated sample is filtered before contacting the treated sample with a detection sensor (e.g., a DNA chip).

In some embodiments, a test sample of appropriate size is collected for use in detecting an assay to provide reliable and sensitive results from the assay. In some examples, a sampling mechanism is used that can efficiently and non-destructively collect test samples to quickly and efficiently extract allergen proteins for detection.

A food coring apparatus 200 such as that shown in fig. 2B may be used to collect a sized portion of the test sample. The food coring apparatus 200 collects samples of appropriate size from which sufficient protein can be extracted for testing. The mass of the sized portion may range from 0.1g to 1g, preferably 0.5 g. In addition, the food coring device 200 may pre-process the collected test sample by cutting, grinding, mixing, grinding, and/or filtering. The pre-treated test sample will be introduced into the homogenization chamber 321 for processing and extraction of allergen proteins.

The collected test samples were processed in extraction/homogenization buffer. In some aspects, the extraction buffer is stored in the homogenization chamber 321 and may be mixed with the test sample by the homogenization rotor 340. In other versions, the extraction buffer may be released into the homogenization chamber 321 from another separate storage chamber. The test sample and extraction buffer will be mixed together by the homogenizing rotor 340 and the homogenized sample. In some embodiments, the extraction buffer is preloaded with a detection agent (e.g., SPN) to allow extraction of the molecule of interest from the test sample to interact with the detection agent.

The extraction buffer may be a universal target extraction buffer that can recover enough target protein from any test sample and can be optimized to maximize protein extraction yield. In some embodiments, a formulation of a universal protein extraction buffer can extract proteins at room temperature and in a minimum time (less than 1 minute). The same buffer can be used during food sampling, homogenization and filtration. The extraction buffer may be a PBS-based buffer containing 10%, 20% or 40% ethanol, or a Tris-based buffer comprising Tris base, 5mM MEDTA and 20% ethanol at pH8.0, or a modified PBS or Tris buffer. In some examples, the buffer may be a HEPES-based buffer. Some examples of modified PBS buffers may include: p + buffer and K buffer. Some examples of Tris-based buffers may include buffer a +, buffer A, B, C, D, E, and buffer T. As a non-limiting example, the extraction buffer may include 20mM EPPS, 2% PEG 8000, 2% F-127(Pluronic), 0.2% Brij-58(pH 8.4). In some embodiments, the extraction buffer may be optimized to increase protein extraction. A detailed description of each modified buffer is disclosed in PCT patent application No. PCT/US 2014/062656; the contents of which are incorporated herein by reference in their entirety.

According to the present disclosure, MgCl is added after the sample is homogenized₂. In some embodiments, after sample homogenization, MgCl₂Solutions (e.g., 30. mu.L of 1M MgCl₂Solution) is added to the homogenization chamber (e.g., chamber 321 in fig. 3F).

In other embodiments, solid MgCl may be used₂Preparation to replace MgCl added during the reaction₂And (3) solution. The solid formulation can be provided as MgCl in a homogenization chamber (e.g., chamber 321 in fig. 3F)₂Lyophilized pellets which are solubilized by the homogenate after filtration, or filter elements deposited or layered in the filter (e.g., filter membrane 420 in fig. 4A and filter assembly 325 in fig. 4A and 6D) (solubilized by the homogenate during filtration), or MgCl deposited on the inner surface of the homogenization chamber 321₂Membranes, or MgCl comprising freeze-dried beads (lyophilized beads) stored in a filtrate chamber (e.g., filtrate chamber 322) or on a separate support₂. In the filter assembly 3In the case of 25, the cotton layer filter (e.g., 412) of the depth filter may be impregnated with the MgCl2 formulation. Regardless of the formulation, MgCl₂Will dissolve in less than 1 minute, preferably in less than 30 seconds, and will contact the treated sample slurry. MgCl₂Can dissolve in about 10 seconds, or about 15 seconds, or about 20 seconds, or about 25 seconds, or about 30 seconds. The solid formulation will release MgCl within this short time₂To reach a final concentration of 30 mM. In some embodiments, the solid MgCl₂The formulation may not break down into powder.

The volume of extraction buffer may be 0.5mL to 3.0 mL. In some embodiments, the volume of extraction buffer can be 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, or 3.0 mL. This volume has been determined to be effective and repeatable over time and in different food matrices.

According to the present disclosure, test samples are homogenized and processed using a homogenizing assembly that has been optimized by high speed homogenization to maximize processing of the test samples.

In some aspects of the present disclosure, the filtration mechanism may be coupled to the homogenizer. The homogenized sample solution is then driven to flow through a filter during processing to further extract allergen proteins and remove particles that may interfere with flow and optical measurements during testing, thereby reducing the amount of other molecules extracted from the test sample. The filtering step may further achieve a uniform viscosity of the sample to control the fluid during the assay. In the case of using a DNA glass chip as a detection sensor, filtration can remove fats and emulsifiers that may adhere to the chip and interfere with optical measurements during testing. In some embodiments, a filtration membrane, such as a cell filter (cell purifier) from CORNING (CORNING, NY, USA) or similar custom embodiments, may be connected to the homogenizer. The filtration process may be a multi-stage arrangement with different pore sizes from the first filter to the second filter, or to the third filter. The filtration process can be adjusted and optimized according to the food substrate to be tested. As a non-limiting example, a filter assembly having a small pore size may be used to capture particles and absorb large amounts of liquid when processing dry food, and therefore, may take longer and higher pressures during the filtration process. In another example, when processing fatty foods, bulk filtration may be performed to absorb fat and emulsifiers. The filtering may further facilitate the removal of fluorescent mist or particles from the fluorescent food (which would interfere with the optical measurement).

The filter may be a simple membrane filter or a module composed of a combination of filter materials (e.g., PET, cotton, sand, etc.). In some embodiments, the homogenized sample may be filtered through a filter membrane or filter assembly (e.g., filter assembly 325 in fig. 4A).

In some aspects of the present disclosure, the sampling procedure can achieve effective protein extraction in less than 1 minute. In one protocol, the digestion (digestion) rate may be less than 2 minutes, which includes food pick up, digestion, and readout. The program may last approximately 15 seconds, 30 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, or 2 minutes.

The extracted allergen proteins may be mixed with one or more detection agents for one or more allergens of interest. The interaction between the allergen protein extract and the detection agent will produce a detectable signal indicative of the presence or absence of one or more allergens in the test sample. As used herein, the term "detection agent" or "allergen detection agent" refers to any molecule capable of interacting with or binding to one or more allergens in a manner that allows for the detection of such allergens in a sample. The detection agent may be a protein-based agent, such as an antibody, a nucleic acid-based agent, or a small molecule.

In some embodiments, the detection agent is a nucleic acid molecule-based Signal Polynucleotide (SPN). SPNs comprise a core nucleic acid sequence that binds a target allergen protein with high specificity and affinity. SPN may be derived from aptamers selected by the SELEX method. As used herein, the term "aptamer" refers to a nucleic acid species engineered by iterative in vitro selection or equivalently SELEX (systematic evolution of ligands by exponential enhancement) design to bind various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organs. The binding specificity and high affinity to the target molecule, the sensitivity and reproducibility at ambient temperature, the relatively low production cost, and the possibility of developing aptamer core sequences capable of recognizing any protein, ensure an efficient but simple detection assay.

SPNs useful as detection agents according to the present disclosure can be aptamers to common allergens (e.g., peanuts, tree nuts, fish, gluten (gluten), milk, and eggs). For example, the detection agent may be an aptamer or SPN as described in applicants' related PCT application publications WO2015066027, WO2016176203, WO2017160616, and WO 2018089391; and U.S. provisional application No. 62/714,102 filed on 3.8.2018; the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the detection agent (e.g., SPN) can be labeled with a fluorescent label. The fluorescent label, fluorophore, may suitably have an excitation maximum in the range of 200nm to 700nm, while the emission maximum may be in the range of 300nm to 800 nm. The fluorophore may also have a fluorescence relaxation time (fluorescence relaxation time) in the range of 1-7 nanoseconds, preferably 3-5 nanoseconds. As one non-limiting example, fluorophores that can be detected at one end of the SPN (terninus) can include boron-dipyrromethene (BODIPY, e.g., BODIPY TMR dye; BODIPY FL dye), fluorescein and its derivatives, rhodamine (rhodamine) and its derivatives, dansyl (dansyls) and its derivatives (e.g., dansylpentamine (dansyl cadeverine)), Texas red, eosin (eosin), cyanine dyes, indocyanine (indocyanine), oxacarbazepine (oxacarbazepine), thiocyanine (thiacarbocyanine), cyanine (merocyanine), squaric acid (squaraine) and its derivatives, thia (derivantivoves seta), thia (seta), squarylium dyes, naphthalene and its derivatives, coumarin and its derivatives, pyridyl oxazole (pyridyloxazole), phenoxy (nitrodiazoline), anthraquinone (oxacarbazepine), and its derivatives, oxazine (oxacarbazone) and its derivatives, Cresol purple (cresyl violet), oxazine 170, proflavine (proflavin), acridine orange, acridine yellow, auramine (auramine), crystal violet, malachite green, porphin (porphin), phthalocyanine (phthalocyanine), bilirubin, tetramethylrhodamine (tetramethylrhodamine), hydroxycoumarin, aminocoumarin (aminocoumarin), methoxycoumarin (methoxycoumarin), cascade blue (cascade blue), pacific blue, pacific orange, NBD, r-Phycoerythrin (PE), red 613; perCP, trured; FluorX, Cy2, Cy3, Cy5 and Cy7, TRITC, X-rhodamine, lissamine rhodamine B, Allophycocyanin (APC), and Alexa fluorescent dyes (e.g., Alexa Fluo 488, Alexa Fluo 500, Alexa Fluo 514, Alexa Fluo 532, Alexa Fluo 546, Alexa Fluo 555, Alexa Fluo 568, Alexa Fluo 594, Alexa Fluo 610, Alexa Fluo 633, Alexa Fluo 637, Alexa Fluo 647, Alexa Fluo 660, Alexa Fluo 680, and Alexa Fluo 700).

In one example, the SPN is labeled with Cy5 at the 5' end of the SPN sequence. In another example, the SPN is labeled with Alexa Fluo 647 at one end of the SPN sequence.

In some embodiments, the SPN for the allergen of interest is pre-stored in the extraction/homogenization buffer in the homogenization chamber 321 (fig. 3B and 3F). The extracted allergen proteins (if present in the test sample) will bind to SPN, forming proteins: an SPN complex. This protein: the SPN complexes can be detected by the detection sensor during the test.

In some embodiments, detection agents for eight major food allergens (i.e., wheat, egg, milk, peanut, tree nut, fish, shellfish, and soy) may be provided as disposables. In one embodiment, the constructs of the detection agent (constructs) may be combined with MgCl₂Stored together or doped with KCl. MgCl₂The construct was kept tightly closed while KC1 opened it slightly for binding.

In some embodiments, the detection sensor is a solid substrate printed with nucleic acids. As used herein, the term "detection sensor" refers to an instrument that can capture a reaction signal (i.e., a reaction signal derived from the binding of an allergen protein and a detection agent), measure the quantity and/or quality of a target, and convert the measurement into a digitally measurable signal.

In some embodiments, the detection sensor is a solid substrate, such as a glass chip, coated with nucleic acid molecules (as referred to herein as a nucleic acid chip or DNA chip). For example, the detection sensor may be a glass chip 333 inserted into the reaction chamber 331 of the present disclosure, or into the chip-based channel 710 in the test cup 300 (fig. 7A). The detection sensor may also be a separate glass chip, for example, made of a glass wafer (wafer) and a calcium sodium glass, or a microwell, or an acrylic glass, or a microchip, or a plastic chip made of COC (cyclic olefin copolymer) and COP (cyclic olefin polymer), or a film-like substrate (e.g., nitrocellulose), the surface of which is covered with nucleic acid molecules.

In some embodiments, the nucleic acid coated chip can include at least one reaction panel and at least two control panels. The reaction panel is printed with nucleic acid probes that hybridize to SPN. As used herein, the term "nucleic acid probe" refers to a short oligonucleotide comprising a nucleic acid sequence complementary to a nucleic acid sequence of SPN. The short complementary sequence of the probe can hybridize to free (free) SPN. When SPN is not bound to the target allergen, SPN can be anchored to the probe by hybridization. When SPN binds to the target allergen to form a protein: SPN complex, protein: the SPN complex prevents hybridization between the SPN and its nucleic acid probe.

In some examples, the probe includes a short nucleic acid sequence complementary to a sequence at the 3' end of the SPN (that specifically binds the allergen protein of interest). In this case, SPN against the allergen protein of interest is provided in the extraction/homogenization buffer. When the sample is processed in the homogenization chamber 321, the target allergen (if present in the test sample) will bind to the SPN and form a protein: an SPN complex. When the sample solution flows to the detection sensor, e.g., the DNA chip 333 or chip-based channel 710 (fig. 7A) in the reaction chamber 331 (fig. 3B), the bound allergen protein prevents the SPN from hybridizing to the complementary SPN probe on the chip surface. Protein: the SPN complexes were washed away and no fluorescent signal was detected. In the absence of the target allergen protein in the test sample, the free SPN will bind to the complementary SPN probes on the chip surface. The fluorescent signal will be detected from the reaction panel (as shown in fig. 13A and 13B).

In some embodiments, the detection sensor (e.g., a chip printed with nucleic acids) further comprises at least two control panels. The control panel is printed with nucleic acid molecules that do not bind to SPNs or proteins (referred to herein as "control nucleic acid molecules"). In some examples, the control nucleic acid molecule is labeled with a fluorescent label.

In some embodiments, nucleic acid probes can be printed to the reaction panel at the center of the glass chip ("unknown"), and control nucleic acid molecules can be printed to both control panels at each side of the reaction panel on the glass chip, as shown in fig. 13A.

In some embodiments, nucleic acid chips (DNA chips) can be prepared by any known DNA printing technique known in the art. In some embodiments, the DNA chip may be prepared by pipetting the nucleic acid solution onto a glass chip using spot pipetting, or by stamping with a wet PDMS stamp containing the nucleic acid probe solution and then pressing the stamp onto a glass slide (glass slide), or by flow with a microfluidic culture chamber.

As one non-limiting example, a glass wafer may be laser diced to produce 10 by 10mm glass "chips". Each chip contains three panels: one reaction panel (i.e., the "unknown" region in the chip shown in FIG. 13A) is sandwiched by two control panels (FIG. 13A). The reaction panel comprises covalently bound short complementary nucleic acid probes bound to SPN against allergen proteins. SPNs are derived from aptamers and modified to contain CY5 fluorophore. In the absence of the target allergen protein, SPNs can bind freely to the probes in the reaction panel, resulting in a high fluorescence signal. SPN: the probe hybridization interface is blocked by the binding of the target protein to the SPN (occlude), resulting in a decrease in the fluorescent signal on the reaction panel. In a detection assay, the reaction panel of the chip faces a small reaction chamber (e.g., reaction chamber 331) that is sandwiched by the inlet and outlet channels (e.g., channel 336 in FIG. 3H) of the cartridge (e.g., cup 300). During food homogenization, SPN in the extraction buffer will bind to the target allergen (if it is present in the sample) to form a protein: an SPN complex. Comprises a protein: the processed sample solution of SPN complexes enters the reaction chamber 331 via an inlet by fluid motion driven by a vacuum pump. The solution then exits via an outlet channel into a waste chamber 323. After exposure to the sample, the reaction panel is then washed, revealing a fluorescent signal whose intensity is correlated to the target allergen concentration.

In some embodiments, the wash buffer is optimized to improve wash efficiency, increase baseline signal, and reduce nonspecific binding. As one non-limiting example, the wash buffer may be an optimized PPB buffer including Pluronic F-127 (e.g., 2% w/v), PEG-8000 (2% w/v), Btij 58 (e.g., 0.2% w/v), and EPPS (e.g., 20mM), pH 8.4.

According to the present disclosure, the two control panels are constantly bright areas on the chip sensor that produce constant signals as background signals 1301 and 1302 (fig. 13B). In addition, the two control panels compensate for laser illumination and/or disposable cassette misalignment. If the cartridge is perfectly aligned, the fluorescent background signals 1301 and 1302 will be equal (as shown in FIG. 13B). If the measured control signals are not equal, a look-up table of correction factors will be used to correct for the unknown signal, which is a function of the cassette/laser misalignment. The final measurement is a comparison of the signal level of the unknown test area 1303 and the control area. The comparison level may be one of the lot-specific parameters (lot-specific parameters) of the test.

Food samples with high background fluorescence measurements from the reaction zone may produce false negative results. A verification method may be provided to adjust the process.

After comparison with these control values (controls), the final fluorescence measurement of the reaction panel and any batch-specific parameters can be analyzed and a report of the results can be provided.

Thus, the light absorption signal and the light scattering signal may also be measured at a baseline level before and/or after injection of the treated food sample. These measurements will provide additional parameters to adjust the detection assay. For example, these signals can be used to look for residual food in the reaction chamber 331 after washing.

In addition to the parameters discussed above, one or more other batch-specific parameters may also be measured. Optimization of these parameters may, for example, minimize the difference (disparity) between the control and unknown signal levels of the chip.

In some embodiments, the monitoring process may be automated and controlled by a software application. The evaluation of the DNA chip and the test sample, the washing process and the final signal measurement can be monitored during the detection assay.

The family of allergens that can be detected using the detection systems and devices described herein include allergens from food, the environment, or from non-human proteins (e.g., house pet dander). Food allergens include, but are not limited to, proteins in legumes (legumes), such as peanuts, peas, lentils and beans (beans), and legume-related plants lupins, tree nuts (such as almonds, cashews, walnuts, Brazil nuts, hazelnuts/hazelnuts, pecans, pistachios, beechnuts, walnuts (butternut), chestnuts, castanets, North American dwarf nuts (chinquapin nuts), coconuts, gingko nuts, lychee nuts, macadamia nuts (macadamia nuts), nangai nuts (nangai nuts) and pines), eggs, fish, shellfish (such as crabs, crayfish, lobsters, shrimp (shrimp) and prawns), mollusks (such as clams, oysters, mussels and scallops), milk, soybeans, wheat, gluten, corn, meat (such as beef, pork, lamb, and chicken), gelatin, sulfites, seeds (such as sesame, poppy seeds and sunflower seeds), And spices (e.g., caraway, garlic, and mustard), fruits, vegetables (e.g., celery), and rice. The allergen may be present in the flour or diet (meal), or in any form of product. For example, seeds from plants (e.g., lupins, sunflowers or poppy) can be used in foods such as seeded bread, or can be ground to make flour for making bread or pastry.

Applications of

The detection systems, devices, and methods described herein contemplate the use of nucleic acid-based detector molecules (e.g., aptamers) to detect allergens in food samples. The portable device allows a user to test a food sample for the presence or absence of one or more allergens. The family of allergens that can be detected using the devices described herein include allergens of leguminous plants (e.g., peanuts), tree nuts, eggs, milk, soybeans, spices, seeds, fish, shellfish, wheat gluten, rice, fruits and vegetables. Allergens may be present in flour or meal. The device is capable of confirming the presence or absence of these allergens and quantifying the amount of these allergens.

In a broad sense, the detection systems, devices, and methods described herein can be used to detect any protein content in a sample in a variety of applications other than food safety, for example, in medical diagnosis of diseases in civilian and battlefield environments, environmental monitoring/control, and military use to detect biological weapons, among others. In a broader application, the detection systems, devices, and methods of the present disclosure can be used to detect any biological molecule to which a nucleic acid-based detection molecule binds. As some non-limiting examples, the detection systems, devices, and methods may be used for in-situ (on the spot) detection of cancer markers, in-field (in-field) diagnostics (exposure chemicals, traumatic head wounds, etc.), third world applications (TB, HIV) testing, etc.), emergency care (stroke markers, head injuries, etc.), and others.

As another non-limiting example, the detection systems, devices, and methods of the present disclosure can detect and identify pathogenic microorganisms in a sample. Pathogens that can be detected include bacteria, yeast, fungi, viruses, and viroid organisms. Pathogens cause diseases in animals and plants; contaminated food, water, soil or other resources; or as a biological agent in the military field. The device is capable of detecting and identifying pathogens.

Another important application includes the use of the detection system, apparatus and method of the present disclosure in medical care, for example, for diagnosing disease, staging disease progression, and monitoring response to certain treatments. As one non-limiting example, the detection devices of the present disclosure can be used to test for the presence or absence, or the quantity, of biomarkers associated with a disease (e.g., cancer) to predict the disease or progression of the disease. The detection systems, devices, and methods of the present disclosure are configured to analyze small amounts of test samples and may be implemented by a user without extensive laboratory training.

Other extended applications outside the field of food safety include field use by military organizations, testing of antibiotics and biopharmaceuticals, environmental testing of products such as pesticides and fertilizers, testing of dietary supplements (dietary supplements) and various food ingredients and additives prepared in bulk (in bulk), such as caffeine and nicotine, and testing of clinical specimens, such as saliva, skin and blood, to determine whether an individual has been exposed to a significant level (significant level) of an individual's allergen.

Experience and scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited by the above description, but rather is defined by the appended claims.

Many possible alternative features are introduced in the course of this description. It is to be understood that such alternative features may be substituted in various combinations that are within the knowledge and judgement of those skilled in the art to obtain different embodiments of the present disclosure.

All or a portion of a patent, publication, internet page, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and where necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Material, or portions thereof, that is described herein as being incorporated by reference, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or otherwise clearly contradicted by context. Claims or descriptions that include an "or" between one or more members (members) are considered satisfied if one or all of the members of the group are present in, used in, or otherwise relevant to a given product or process unless otherwise indicated herein as opposed to or otherwise apparent from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which multiple or an entire group of members of the group exist, are used, or are otherwise relevant to a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, then the term "consisting of … … (governing of)" is also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value or sub-range within the stated range, up to one tenth of the unit of the lower value of the range, in different embodiments of the invention, unless the context clearly dictates otherwise.

Furthermore, it is to be understood that any particular embodiment of the disclosure that falls within the scope of the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if such exclusion is not explicitly set forth herein. For whatever reason, any particular embodiment of a composition of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of manufacture; any method of use; etc.) can be excluded from any one or more claims, whether or not related to the presence of prior art.

It is understood that the words which have been used are words of description rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

While the present disclosure has been described in some length and with respect to features of several described embodiments, it is not intended to limit the disclosure to any such specific embodiment or embodiments or any particular embodiment, but rather should be construed with reference to the appended claims so as to provide the broadest interpretation of such claims with respect to the prior art and, therefore, to effectively encompass the intended scope of the invention.

Examples of the invention

Example 1: testing of Filter Material and Filter efficiency

Various filter materials and combinations thereof were tested for filtration efficiency and impact on signal measurement, e.g., loss of detection agent (SPN). Commercially available filter materials were tested, such as membranes (PES, glass fiber, PET, PVDF, etc.), cotton, sand, mesh and silica.

A filter comprising a combination of different filter materials is assembled. In one example, the filter assembly consists of a glass filter with a pore size of 1 μm and cotton. The cotton depth filter and the paper filter are configured to sequentially filter the sample. The filter assembly was tested for filtering different food substrates. The recovery of protein and SPN during the filtration process was measured. Various cotton volumes were used to construct the depth filter, and the cotton depth filter was combined with a membrane filter. These filter assemblies were tested for filtration efficiency and SPN recovery. In one study, 0.5g food samples were collected and homogenized in 5ml EPPS buffer (pH8.4) (tween 0.1%) and the homogenized food samples were then incubated with 5nM SPN (signal polynucleotide) labeled with Cy5 for allergen proteins. After incubation, a portion of the mixture was passed through a filter assembly and the recovery of protein and SPN was measured and compared to the measurement before filtration.

The filter is further tested and optimized to ensure the efficiency of the filtration and to avoid significant SPN loss. In addition to testing different filter materials and combinations thereof, other parameters such as pore size, filter area (e.g., surface area/diameter of depth filter, height), filter volume required to drive the filtration process, filter time and pressure, etc. were also tested and optimized for various food substrates.

In one study, bleached cotton balls were used to assemble depth filters with different filtration volumes. Cotton filters with different widths (i.e., diameters) and height ratios are constructed; the width to height ratio of each mold is in the range of about 1:30 to about 1: 5. The cotton depth filter was then tested for filtration efficiency at different food mass and buffer volumes. In another study, these types of cotton filters had a pore size of 1 μm and a filtration area of about 20mm²Assembled together. Various food samples were homogenized and filtered through each filter assembly using different volumes of buffer. The filtrates were collected and the recoveries at each condition were compared.

In another study, food samples were spiked with 50ppm peanuts or without spiking. The spiked sample is homogenized, for example, using a rotor 340 (e.g., as shown in fig. 3B and 3C), and the extract is mixed with SPNs that specifically bind peanut allergens. SPN contains a Cy5 tag at the 5' end of the sequence. The mixture is filtered through a depth filter (for example, a depth filter made of cotton) and a membrane filter (pore size: 1 μm). The fluorescence signal is measured and compared to the measurement of the pre-filtered mixture.

In a separate study, several parameters of each filter assembly were tested and measured, including the pressure and time required for filtration, binding of proteins and nucleic acids, washing efficiency, and assay compatibility and sensitivity. Assay compatibility was measured as baseline intensity.

Example 2: MgCl₂Preparation

After homogenization of the samples in extraction buffer, several solid MgCl₂The formulations were tested to replace MgCl₂And (4) adding the solution. The following characteristics of each formulation tested were evaluated: (1) the time of dissolution; (2) dissolved MgCl₂(ii) a final concentration of (d); (3) the effect of the additive in the formulation on the detection assay; (4) the dissolution does not need stirring; (5) there is no crushing into powder and no blocking of the outlet of the homogenizing chamber.

Freeze-dried MgCl₂Preparation

34 kinds of MgCl₂The formulations were freeze-dried in 1.5mL Eppendorf tubes and tested for dissolution time, mechanical stability, exposure to extraction buffer for 10 seconds without stirring, and other functions. 2 the formulation dissolves rapidly and does not form a powder. Several MgCl types₂The formulation was exposed to the extraction buffer without stirring for 10 seconds and the magnesium content in the recovery buffer was determined by the BioVision magnesium assay and the assay described herein. The assay results showed freeze-dried MgCl including maltodextrin and hydroxyethyl cellulose (HEC)₂The highest strength of SPN in the buffer is given by the formulation (table 1) as shown in fig. 16A.

MgCl₂As a filtering component

MgCl₂The formulation (table 1) was deposited on a cotton filter and dried at 60 ℃. The extraction buffer was pulled through the cotton filter at a vacuum of 1 psi. The percentage of magnesium recovered in the filtrate was measured by the BioVision colorimetric magnesium assay. MgCl including maltodextrin and hydroxyethyl cellulose (HEC)₂Formulations (Table 1) with MgCl₂Recovered in solution and MgCl on filter₂Comparison was made (fig. 16B).

MgCl₂As a film (film)

10 different MgCl₂The formulation is deposited on a polystyrene support and cured. Dissolution time was measured and all formulations were dissolved within 10 seconds. The results show that none of the formulations have strong adhesion to the polystyrene support.

Table 1: MgCl₂Ingredients of the formulation

Based on the test results, several fast dissolving MgCl were selected₂Solid formulations (as shown in table 2). The dissolution time of the filter deposit depends on the flow rate. When the fastest flow rate was tested, the solid formulation was dissolved within 10 seconds (as shown in table 2).

Table 2: fast dissolving and mechanically robust (robust) solid MgCl₂Preparation

Claims (83)

1. An assembly for detecting a molecule of interest in a sample, comprising:

a sample processing cartridge configured to receive a sample to be processed in a state allowing interaction of the molecule of interest with a detection agent;

a detector unit configured to accept the sample processing cartridge in a configuration that allows detection of interaction of the molecule of interest with the detection agent by a detection mechanism housed by the detector unit, wherein the interaction triggers a visual indication on the detector unit that the molecule of interest is detected,

wherein the visual indication is an image captured by processing of the interaction of the molecule of interest and the detection agent.

2. The assembly of claim 1, wherein the molecule of interest is an allergen.

3. The assembly of claim 1 or 2, wherein the detection agent is an antibody or a variant thereof, a nucleic acid molecule or a variant thereof, or a small molecule.

4. The assembly of claim 3, wherein the detection agent is a nucleic acid molecule or variant thereof.

5. The assembly of claim 4, wherein the nucleic acid molecule is an aptamer-derived Signal Polynucleotide (SPN) comprising a nucleic acid sequence that binds to the molecule of interest.

6. The assembly of any one of claims 1 to 5, wherein the sample processing cartridge comprises:

a homogenizer configured to produce a homogeneous sample, thereby releasing the molecule of interest from the matrix of the sample into an extraction buffer in the presence of a detection agent;

a plurality of separate chambers including a homogenization chamber, a filtrate chamber, and a detection chamber;

a first conduit to transfer the homogeneous sample and a detection agent through a filter system to provide a filtrate containing the molecule of interest and the detection agent; and

a second conduit to transfer the filtrate into a detection chamber having a window;

wherein a detection mechanism of the detector unit analyzes the detection chamber through the window to identify interactions of the molecule of interest with a detection agent in the detection chamber.

7. The assembly of claim 6, wherein the homogenizer comprises a rotor, and wherein the rotor is powered by a motor located in the detector unit, wherein the motor is functionally coupled to the homogenizer when the sample processing cartridge is accepted by the detector unit.

8. The assembly of claim 6 or 7, wherein the sample processing cartridge further comprises: a chamber holding a wash buffer for washing the detection chamber; and a waste chamber for receiving the effluent contents of the detection chamber after washing.

9. The assembly of claim 8, wherein the sample processing cartridge further comprises: a rotary valve system for controlling transfer of the homogeneous sample to the filter system, for transferring the filtrate to the detection chamber, for transferring the wash buffer to the detection chamber, and for transferring the contents of the detection chamber to the waste chamber.

10. The assembly of claim 9, wherein the rotary valve system is further configured to provide a closed position to prevent fluid movement in the sample processing cartridge.

11. The assembly of any one of claims 6 to 10, wherein the detection chamber comprises a transparent substrate having detection probe molecules immobilized thereon, the detection probes configured to probe interact with the detection agent, wherein interaction of the molecule of interest with the detection agent prevents probe interaction of the detection agent with the detection probes.

12. The assembly of claim 11, wherein the transparent substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

13. The assembly of claim 11, wherein the transparent substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

14. The assembly of claim 12 or 13, wherein the detection probes and control probes are immobilized on the transparent substrate in a checkerboard pattern.

15. The assembly of claim 14, wherein the detection agent comprises an optically detectable moiety that is activated upon the probe interaction.

16. The assembly of claim 15, wherein the optically detectable moiety is a fluorescent moiety.

17. The assembly of any one of claims 11 to 16, wherein the detection mechanism housed by the detector unit is a fluorescence detection system having an LED for exciting fluorescence, the fluorescence detection system being configured to detect fluorescence emission signals and background signals when the probe interaction is performed and subject to fluorescence excitation.

18. The assembly of claim 17, wherein the detection mechanism comprises a plurality of optical elements placed in a linear or folded arrangement within a stepped bore in the detector unit.

19. The assembly of claim 17, wherein the detector unit further comprises: a camera-based detector for capturing the reaction on the transparent substrate and analyzing the fluorescence emission signal and background signal to identify the probe interaction and communicate the identity of the molecule of interest, or the source of the molecule of interest, to the visual indication so as to inform an operator of the assembly whether the molecule of interest or the source of the molecule of interest is present in the sample.

20. The assembly of any one of claims 11 to 19, wherein the transparent substrate comprises a plurality of different detection probes for detecting a plurality of different detection agents configured to provide a plurality of different interactions with different molecules of interest in the sample.

21. The assembly of claim 20, wherein the transparent substrate further comprises a fluidic panel associated with the probes for transferring a filtrate comprising the molecule of interest and the detection agent to contact the detection probes and control probes.

22. The assembly of any one of claims 1 to 21, further comprising: a sampler comprising a hollow tube having a cutting edge for cutting a source to create and hold the sample in the hollow tube; and a plunger for pushing the sample out of the hollow tube into a port in the sample processing cartridge.

23. An analysis cartridge for detecting a molecule of interest in a sample, comprising:

(a) a first compartment having a homogenizer for receiving a sample and processing the sample, the homogenizer configured to produce a homogenized sample, thereby releasing the molecule of interest from a matrix of the sample into an extraction buffer in the presence of a detection agent and allowing the molecule of interest in the sample to interact with the detection agent;

(b) a conduit to transfer the homogeneous sample and the detection agent through a filtration system to provide a filtrate containing the molecule of interest and the detection agent;

(c) a second compartment for contacting the filtrate containing the molecule of interest and the detection agent by means of a detection probe; the second compartment comprises a transparent substrate comprising a fluidic channel and a detection chip region having detection probes immobilized thereon, the detection probes configured to perform a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from performing a probe interaction with the detection probes;

(d) a rotary valve system configured to regulate transfer of the homogeneous sample and the detection agent through the filtration system, transfer of the filtrate to the second compartment, and transfer of wash buffer to the second compartment, and effluent contents from the second compartment to a waste chamber;

(e) a compartment for holding a wash buffer for washing the detection zone; and

(f) a waste chamber for receiving the effluent contents of the detection chamber.

24. The analysis cartridge of claim 23, wherein the second compartment comprises a window through which a detection mechanism of a detector unit analyzes a detection reaction to identify interaction of the molecule of interest with the detection agent in the second compartment.

25. The analysis cartridge of claim 24, wherein the detection region of the transparent substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

26. The analysis cartridge of claim 24, wherein the substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

27. The assay cartridge of any of claims 23-26, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the molecule of interest.

28. The analysis cartridge of claim 27, wherein the nucleic acid-based detection agent is a Signal Polynucleotide (SPN) derived from an aptamer comprising a nucleic acid sequence that binds to the molecule of interest.

29. The assay cartridge of claim 28, wherein the detection agent comprises an optically detectable fluorescent moiety that is activated when the probe interaction is performed.

30. The assay cartridge of any of claims 25-29, wherein the detection probe is a nucleic acid molecule comprising a nucleic acid sequence complementary to a nucleic acid sequence of the detection agent.

31. The analysis cartridge of any of claims 23-30, wherein the substrate is a glass chip, or a plastic chip, or a film chip.

32. The analysis cartridge of claim 31, wherein the filter system consists of a body filter comprising a cotton volume and a membrane filter.

33. The analysis cartridge of claim 32, wherein the cartridge further comprises: a plurality of fluid flow paths for transferring the homogeneous sample to the filter system, for transferring the filtrate to the transparent substrate, for transferring the wash buffer to the detection chamber, and for transferring the contents of the detection chamber to the waste chamber.

34. The analysis cartridge of any of claims 23-33, wherein the rotary valve system is further configured to provide a closed position to prevent movement of fluid in the cartridge.

35. The analysis cartridge of any of claims 23-34, wherein the molecule of interest is an allergen.

36. A test cup assembly for processing a sample to a state that allows detection of a molecule of interest in the sample, comprising:

a top cover for sealing the test cup and providing an identification label;

a body portion for receiving and processing the sample to a state that allows interaction of the molecule of interest in the sample with a detection agent, the body portion comprising:

(i) a first compartment having a homogenizer for homogenizing the sample using an extraction buffer to extract the molecules of interest, thereby releasing the molecules of interest from the matrix of the sample into the extraction buffer and interacting with a detection agent present in the extraction buffer;

(ii) a conduit to transfer a homogeneous sample containing the molecule of interest and a detection agent through a filter system to provide a filtrate containing the molecule of interest and the detection agent;

(iii) a chamber for holding a wash buffer;

(iv) a waste chamber for receiving and storing effluent contents after washing the molecules of interest and detection agents; and

(v) a rotary valve system for controlling fluid movement inside the test cup assembly;

a transparent substrate comprising a plurality of fluidic channels and a detection region having detection probes immobilized thereon, the detection probes configured to perform a probe interaction with the detection agent, wherein interaction of the molecule of interest with the detection agent prevents the detection agent from performing the probe interaction with the detection probes; and

a bottom cover for sealing the test cup and providing an interface to connect the test cup to a detector unit for operational detection; the bottom cover includes a transparent window that aligns with the detection zone of the transparent substrate when the test cup is assembled.

37. The test cup assembly of claim 36, wherein the cup top cover includes a port for receiving the sample, and at least one vent filter to allow air to enter.

38. The test cup assembly of claim 35 or 36, wherein the detection mechanism of the detector unit analyzes the interaction of the molecule of interest with the detection agent and the interaction between the detection agent and the detection probes.

39. The test cup assembly of claim 38, wherein an exterior of the bottom cover includes a plurality of ports for connecting a plurality of motors housed in the detector unit to operate the homogenizer, the rotary valve system, and the flow of fluid in the test cup assembly.

40. The test cup assembly of claim 39, wherein said filter system is comprised of a coarse filter, a depth filter, a membrane filter, and wherein said filter system further comprises a filter cover.

41. The test cup assembly of any one of claims 36 to 40, wherein the cup bottom comprises a plurality of compression coil springs supporting the rotary valve system.

42. The test cup assembly of claim 41, wherein the filter cover is connected to the rotary valve.

43. A test cup assembly according to any one of claims 36 to 42, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that specifically binds to the molecule of interest.

44. The test cup assembly of claim 43, wherein the detection agent is a Signal Polynucleotide (SPN) comprising an aptamer having a nucleic acid sequence that binds to the molecule of interest.

45. The test cup assembly of claim 44, wherein said detection probe is a nucleic acid molecule comprising a nucleic acid sequence complementary to a sequence of said detection agent.

46. The test cup assembly of claim 45, wherein the detection region of the transparent substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

47. The test cup assembly of claim 45, wherein the detection area of the transparent substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

48. A test cup assembly according to any one of claims 46 to 47, wherein the detection area of the transparent substrate further comprises one or more fiducial marks.

49. The test cup assembly of any one of claims 36 to 48, further comprising a data chip.

50. A system for detecting the presence or absence of a molecule of interest in a sample, comprising:

a sampler for collecting a sample suspected of containing the molecule of interest;

a disposable analysis cartridge configured for processing the sample, thereby allowing interaction of the molecule of interest in the sample with a detection agent; and

a detection device configured for operating a detection test and measuring and visualizing a signal from a binding interaction between the detection agent and the molecule of interest present in the sample.

51. The system of claim 50, wherein the sampler is a distal to proximal food corer comprising a plunger, a skirt, and a corer, wherein the proximal end of the corer comprises a cutting edge for cutting the test sample.

52. The system of claim 51, wherein the disposable analysis cartridge comprises:

(i) a sample processing chamber having a homogenizer configured to homogenize the sample with an extraction buffer in the presence of a detection agent, thereby allowing an allergen of interest in the sample to interact with the detection agent;

(ii) a filter system configured to provide a filtrate comprising the allergen of interest and the detection agent;

(iii) a single transparent substrate comprising a plurality of fluidic channels and detection regions having detection probe molecules immobilized thereon; the detection probe is configured to perform a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from performing a probe interaction with the detection probe;

(iv) a detection chamber having an optical window;

(v) a chamber holding a wash buffer for washing the substrate and the detection chamber;

(vi) a waste chamber for receiving and storing the effluent contents of the detection chamber after washing;

(vii) a rotary valve system and a plurality of conduits configured to transfer a homogeneous sample and detection agent through the filter system to transfer the filtrate to the detection chamber, the wash buffer to the detection chamber, and the effluent contents from the detection chamber to the waste tank, and

(viii) an air flow system configured to regulate air pressure and flow rate in the cassette.

53. The system of claim 52, wherein the filter system comprises a main body filter consisting of a coarse filter and a depth filter, and a membrane filter.

54. The system of claim 53, wherein the filter system further comprises a filter hood connected to the rotary valve system.

55. The system of any one of claims 50 to 54, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that specifically binds to the molecule of interest.

56. The system of claim 55, wherein the detection agent is a Signaling Polynucleotide (SPN) derived from an aptamer comprising a nucleic acid sequence that specifically binds to the molecule of interest.

57. The system of any one of claims 50-56, wherein the analysis cartridge further comprises MgCl₂Freeze-dried beads.

58. The system of any one of claims 50 to 57, wherein the detection zone of the transparent substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

59. The system according to any one of claims 50 to 57, wherein the detection zone of the transparent substrate further comprises two optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

60. The system of any one of claims 58 to 59, wherein the transparent substrate is selected from the group consisting of a glass chip, silica, agarose particles, acrylic glass, microwells, and microchips.

61. The system of claim 53, wherein the filtration membrane comprises at least one membrane selected from the group consisting of a nylon membrane, PE, PET, PES (polyethersulfone) membrane, glass fiber membrane, polymer membrane, Mixed Cellulose Ester (MCE) membrane, cellulose acetate membrane, PTFE membrane, polycarbonate membrane, PCTE (polycarbonate) membrane, and PVDF (polyvinylidene fluoride) membrane.

62. The system of claim 50, wherein the detection device comprises an external housing configured to provide support for a plurality of components of the detection device; the plurality of components integrated for operational testing, comprising:

(i) a motor for driving and controlling the homogenization of the sample,

(ii) for controlling the motor of the valve system,

(iii) a pump for driving and controlling the flow of the fluid,

(iv) an optical system for detecting a fluorescent signal,

(v) means for converting and digitizing the fluorescence signal,

(vi) a display window for receiving the detected signal and indicating the presence or absence of the allergen in the test sample, an

(vii) A power source.

63. The system of claim 62, wherein the optical system comprises: the excitation type optical device consists of a Light Emitting Diode (LED), a collimating lens, a filter and a focusing lens; the emission type optical device consists of a focusing lens, two emission filters, one or more converging lenses and an aperture; and a camera.

64. The system of claim 63, wherein the individual transparent substrates are aligned with the optical system of the device via an optical window of the detection chamber.

65. A signal detection unit for detecting and measuring a fluorescent signal, comprising:

a motor module for operating the sample processing unit when the sample processing unit is connected to the signal detection unit;

a pump module for controlling fluid flow inside the sample processing cell;

an optical system for detecting a fluorescence signal from the sample processing unit; and

a power source.

66. The signal detection unit of claim 65, wherein the unit further comprises a docking surface for receiving the sample processing unit.

67. The signal detection unit of claim 66, wherein the optical system comprises: the excitation type optical device consists of a Light Emitting Diode (LED), a collimating lens, a filter and a focusing lens; and emission optics consisting of a focusing lens, two emission filters, one or more converging lenses and an aperture; and a camera.

68. The signal detection unit of any one of claims 65 to 67, wherein the unit further comprises: a digital processing unit for processing the fluorescence signal; and the display window is used for displaying the detection result.

69. The signal detection unit of claim 68, wherein the motor module comprises a motor driving a valve system and a motor driving a homogenizer of the sample processing unit,

wherein the motor driving the valve system includes: a dc gear motor having two optical sensors: an output optical sensor and a direct drive shaft optical sensor; and the microcontroller comprises an output connector, an encoder wheel, a direct drive motor shaft and a direct drive shaft encoder wheel.

70. A method for detecting the presence or absence of a molecule of interest in a sample, comprising:

(a) collecting a sample and treating the sample in an extraction buffer in the presence of a detection agent, thereby allowing the molecule of interest to interact with the detection agent;

(b) filtering the treated sample containing the molecule of interest and the detection agent;

(c) contacting the filtrate with a substrate having detection probes immobilized thereon; the detection probe is configured to perform a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from performing a probe interaction with the detection probe;

(d) washing unbound compounds from the substrate with a wash buffer;

(e) measuring a fluorescent signal from the substrate; and

(f) detecting the presence or absence of the molecule of interest in the sample.

71. The method of claim 70, wherein the substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

72. The method of claim 70, wherein the substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

73. The method of any one of claims 70 to 72, wherein the detection agent is a nucleic acid molecule comprising a nucleotide sequence that specifically binds to the molecule of interest.

74. The method of claim 73, wherein the detection probe is a nucleic acid sequence comprising a nucleotide sequence complementary to a sequence of the detection agent or a portion thereof, and the nucleic acid sequence hybridizes to the sequence of the detection agent when the sequence of the detection agent does not interact with the molecule of interest.

75. The method of claim 74, wherein the substrate is a plastic chip.

76. The method of any one of claims 70-75, wherein the treated sample and the detection agent are filtered by a filter assembly comprising a strainer, a depth filter, and a membrane filter.

77. The method of claim 76, wherein the depth filter is a cotton depth filter.

78. The method of claim 76 or 77, wherein the detection agent is formulated as MgCl₂And (4) pills.

79. The method of claim 70, wherein the molecule of interest is an allergen.

80. The method of claim 79, wherein the sample is a food sample.

81. A kit comprising a test cartridge according to any one of claims 23 to 35 or a test cup assembly according to any one of claims 36 to 49 and instructions for using the test cartridge or the test cup to test for the presence of a molecule of interest in a sample.

82. The kit according to claim 81, further comprising: a sampler for collecting a sample while testing for the presence of the molecule of interest in the sample.

83. The kit according to claim 82, further comprising: a detection unit for operating the test cartridge or the test cup assembly when testing for the presence of the molecule of interest in the sample.

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